Antibodies against anthrax protective antigen

ABSTRACT

A highly efficient method for generating human antibodies using recall technology is provided. In one aspect, human antibodies which are specific to the anthrax toxin are provided. In one aspect, human peripheral blood cells that have been pre-exposed to anthrax toxin are used in the SCID mouse model. This method results in high human antibody titers which are primarily of the IgG isotype and which contain antibodies of high specificity and affinity to desired antigens. The antibodies generated by this method can be used therapeutically and prophylactically for preventing or treating mammals exposed to anthrax. Thus, in one embodiment, a prophylactic or therapeutic agent used to counter the effects of anthrax toxin, released as a mechanism of bioterrorism, is provided. In one embodiment, a formulation and method for preventing and/or treating anthrax infection comprising a binding agent that prevents the assembly of the PA63 heptamer is also provided. Methods for diagnosis and methods to determine anthrax contamination are also described.

RELATED APPLICATIONS

This application is a continuation of United States application No.[Atty Docket No. AVANIR.149CP1], filed Jan. 21, 2005, and is acontinuation-in-part of PCT Application No. PCT/US2003/36555, filed Nov.14, 2003 under 35 U.S.C. § 120, and also claims benefit to U.S.provisional application Ser. No. 60/538,721, filed Jan. 23, 2004 andSer. No. 60/562,421, filed Apr. 15, 2004, all herein incorporated byreference.

GOVERNMENT INTEREST

This invention was made, in part, with the support of the United StatesGovernment under the following grants: CCAT # 52109B 7806, NIAID #R43AI52901-1A1, and NIAID # R43 AI 158458-01. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to fully human monoclonalantibodies, method of making same, and their use in preventive andtherapeutic applications for anthrax. In one aspect, antibodies thathave binding specificity for anthrax protective antigen (PA) toxin areprovided.

2. Description of the Related Art

Anthrax is a zoonotic soil organism endemic to many parts of the world.The Bacillus anthracis organism was one of the first biological warfareagents to be developed and continues to be a major threat in thisregard. The Centers for Disease Control and Prevention (CDC) hasemphasized that the United States faces a new wave of terrorism, in theform of a biological attack. For example, in late 2001, Bacillusanthracis spores were intentionally distributed through the postalsystem, causing 22 cases of anthrax, including 5 deaths. Anthrax is akey toxin that can be employed by terrorists to debilitate a nation.

Although vaccine strains have been developed for anthrax, currentlythere are concerns regarding their efficacy and availability. Afterinhalation by mammals, Bacillus anthracis spores germinate in thealveolar macrophages, then migrate to lymph nodes where they multiplyand enter the bloodstream. The vegetative bacteria excrete thetripartite exotoxin that is responsible for the etiology of the disease.Virulent strains of Bacillus anthracis secrete a set of three distinctantigenic protein components: protective antigen (PA), edema factor(EF), and lethal factor (LF). PA can bind either LF or EF, forminglethal toxin (LeTx) or edema toxin (EdTx). Collectively these two toxinsare seen as a complex exotoxin called anthrax toxin. Each component ofthe toxin is a thermolabile protein with a molecular weight exceeding 80kDa. EF is a calmodulin dependent adenylate cyclase that is responsiblefor the edema seen in anthrax infections. LF is a zinc-metalloproteasethat is needed for the lethal effect of the anthrax toxin onmacrophages. It is believed that PA contains the binding domain ofanthrax toxin, which binds to recently identified receptors on the cellsurface called collectively anthrax toxin receptors (ATRs) and allowstranslocation of LF or EF into the cell by endocytosis.

Evidence that the hu-PBL-SCID system (severe combined immunodeficient(SCID) mice engrafted with human peripheral blood leukocytes) can beused to obtain recall antibody responses dates from the originalpublication of the method by Mosier and co-workers. Mosier et al.,Nature 335:256 (1988), herein incorporated by reference. In that report,tetanus toxoid was administered to human PBL engrafted mice, and humanantibodies to tetanus were found in the serum post-immunization. Sincethis original report, many investigators have used the hu-PBL-SCIDsystem to examine aspects of the human recall antibody response tomultiple antigens. See, for example, Nonoyama, S. et al., J. Immunol.,151:3894 (1993); Walker, W. et al., Eur. J. Immunol., 25:1425 (1995);Else, K. J., and Betts. C. J., Parasite Immunology 19:485 (1997), allherein incorporated by reference. However, reports describing thegeneration of useful monoclonal antibodies from such engrafted mice havebeen sporadic. Duchosal, M. A. et al., Letters To Nature: 258 (1991);Satoh, N. et al., Immunology Letters 47:113 (1995); Uchibayashi, N. etal., Hybridoma 14:313 (1995). Nguyen, H. et al., Microbiol. Immunol.41:901 (1997); Coccia, M. A and P. Brams, Amer. Assoc. Immunologists:5772 (1998); and Smithson, S. L. et al., Molecular Immunology 36:113(1999), all herein incorporated by reference.

SUMMARY OF THE INVENTION

There remains a need for an effective method to produce human monoclonalantibodies that are specific to a particular antigen. Moreover, a needfor a fully human monoclonal antibody specific to the anthrax toxinstill remains. Accordingly, in a preferred embodiment of the presentinvention, a novel anti-anthrax antibody that is fully human isprovided. This new fully human anthrax antibody can be administered to amammal to confer immunity to that mammal. Because the antibody isadministered directly (instead of the antigen), this technique is called“passive immunization.” Thus, in one embodiment of the presentinvention, a method of passively immunizing a mammal is provided usingone or more novel antibodies of the invention. Several embodiments ofthe present invention are also used to treat mammals that have beenexposed to anthrax, thereby preventing the toxic effects of anthraxpost-exposure and/or reducing the severity of the illness.

In one embodiment of the present invention, a composition and method tocounter the effects of anthrax toxin that is released as a mechanism ofbioterrorism are provided. In one embodiment, a prophylactic treatmentin the form of passive immunization is provided. In one embodiment,antibodies to anthrax are administered to a mammal to prevent anthraxinfection and/or to treat anthrax infection. One advantage of a passiveimmunization strategy is that it may useful in conferring immediate tomedium-term protection, and can also have benefits for non-immunizedpatients who seek treatment after the point at which antibiotic therapyalone is ineffective. Casadevall, A., Emerging Infectious Diseases, 8:8(2002); Maynard, J. A et al., Nature Biotechnology, 20:597 (2002),herein incorporated by reference. Passive immunization, according tosome embodiments of the invention, can confer short-term, long-termand/or permanent protection to recipients.

In some embodiments of the present invention, antibodies that bind tothe PA component of the tripartite anthrax exotoxin are provided. Theseantibodies will provide protection either as single agents or combinedin a cocktail. A method to generate a series of fully human anti-anthraxPA toxin antibodies is also provided.

In one embodiment, a fully human monoclonal antibody, or fragmentthereof, is disclosed which specifically recognizes at least a portionof an anthrax exotoxin. In one variation, the portion of an anthraxexotoxin is selected from the group consisting of protective antigen(PA), lethal factor (LF) and edema factor (EF). In one embodiment, theantibody recognizes only PA.

In one embodiment, a fully human monoclonal antibody or fragment thereofthat recognizes at least a portion of an anthrax exotoxin and comprisesan amino acid sequence selected from the group consisting of: SEQ ID 2,SEQ ID 4, SEQ ID 6, SEQ ID 8, SEQ ID 10, SEQ ID 12, SEQ ID 14, and SEQID 16 is provided. In one embodiment, two or more antibodies areprovided. In one embodiment, an antibody comprising SEQ ID 2 and SEQ 4is provided. In another embodiment, an antibody comprising SEQ ID 6 andSEQ 8 is provided. In a further embodiment, an antibody comprising SEQID 2, SEQ 4, SEQ ID 6 and SEQ 8 is provided.

In one embodiment, a fully human monoclonal antibody encoded at least inpart by a polynucleotide comprising a nucleotide sequence selected frQmthe group consisting of: SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID9, SEQ ID 11, SEQ ID 13, and SEQ ID 15 is provided. In one embodiment,two or more antibodies are provided. In another embodiment, a hybridomacomprising one or more of the following nucleotide sequences: SEQ ID 1,SEQ ID 3, SEQ ID 5, SEQ ID 7, SEQ ID 9, SEQ ID 11, SEQ ID 13, and SEQ ID15 is provided.

In one embodiment, one or more of the following antibodies, or fragmentsthereof, are provided: antibody 21D9, antibody 22G12, antibody 1C6, andantibody 4H7. In a preferred embodiment, two or three antibodies areprovided. For example, in one embodiment, 21D9 and 1C6 are provided. Inseveral embodiments, the administration of two or more antibodies showsenhanced or synergistic effects. Chemical modifications, mutations, andother variants of these antibodies are also provided, including but notlimited to 21D9.1 and 22G12.1. Methods of making and using theantibodies, or fragments thereof, are also provided.

In a further embodiment, a pharmaceutical composition for passivelyimmunizing a mammal against anthrax, wherein the pharmaceuticalcomposition comprises one or more of the fully human monoclonalantibodies, or fragments thereof, described above, is provided. In oneembodiment, the mammal has not been previously exposed to anthrax.

In one embodiment, a pharmaceutical composition for treating a mammalexposed to anthrax, wherein the pharmaceutical composition comprises oneor more of the fully human monoclonal antibodies, or fragments thereof,described above, is provided.

In one embodiment, the pharmaceutical composition comprises twodifferent fully human monoclonal antibodies, or fragments thereof. Insome embodiments, the pharmaceutical composition comprises a monoclonalantibody that comprises less than 100% human protein sequences (forexample, a humanized antibody or a partially human antibody). In afurther embodiment, the pharmaceutical composition further comprisesAnthrax Vaccine Adsorbed (AVA) or recombinant protective antigen (rPA).In one embodiment, the pharmaceutical composition comprises anantibiotic.

In another embodiment, the immunoglobulin or fragment thereof comprisesan immunoglobulin heavy chain variable region comprising FR1, CDR1, FR2,CDR2, FR3, CDR3, and FR4; wherein the variable region is comprised ofthe amino acid sequence shown in FIG. 5. A “CDR” is a complementaritydetermining region. An “FR” is a frame work region. In one embodiment,an antibody comprising CDR1, CDR2, and CDR3 shown in SEQ ID 2 isprovided.

In another embodiment, the immunoglobulin or fragment thereof comprisesan immunoglobulin light chain variable region comprising FR1, CDR1, FR2,CDR2, FR3, CDR3, and FR4; wherein the variable region is comprised ofthe amino acid sequence shown in FIG. 6. In one embodiment, an antibodycomprising heavy chain CDR1, CDR2, and CDR3, all as shown in FIG. 5, isprovided. In another embodiment, an antibody comprising light chainCDR1, CDR2, and CDR3, all as shown in FIG. 6, is provided. In apreferred embodiment, an antibody comprising the heavy and light chainCDRs shown in FIG. 5 and FIG. 6 is provided.

In one preferred embodiment of the present invention, the fully humanimmunoglobulin or fragment thereof is a single chain that recognizes atleast a portion of an anthrax exotoxin.

In one preferred embodiment, a fully human immunoglobulin or fragmentthereof is disclosed, that recognizes at least a portion of an anthraxexotoxin, wherein the immunoglobulin or fragment thereof comprises animmunoglobulin heavy chain comprising at least a portion of the aminoacid sequence shown in FIG. 8 and/or an immunoglobulin light chaincomprising at least a portion of the amino acid sequence shown in FIG.8. In one embodiment, the antibody comprises an immunoglobulin heavychain comprising at least a portion of the amino acid sequence shown inFIG. 9 and/or an immunoglobulin light chain comprising at least aportion of the amino acid sequence shown in FIG. 9. In one embodiment,the antibody comprises an immunoglobulin heavy chain comprising at leasta portion of the amino acid sequence shown in FIG. 10 and/or animmunoglobulin light chain comprising at least a portion of the aminoacid sequence shown in FIG. 10.

In one preferred embodiment, a fully human immunoglobulin or fragmentthereof is disclosed, that recognizes at least a portion of an anthraxexotoxin, wherein the immunoglobulin or fragment thereof comprises animmunoglobulin light chain comprising at least one complementarydetermining region selected from the group consisting of CDR1, CDR2 andCDR3; wherein the CDR1 is comprised of the amino acid sequence, as shownin FIG. 6; the CDR2 is comprised of the amino acid sequence, as shown inFIG. 6; and the CDR3 is comprised of the amino acid sequence, as shownin FIG. 6.

In another preferred embodiment, a fully human immunoglobulin orfragment thereof is disclosed, that recognizes at least a portion of ananthrax exotoxin, wherein the immunoglobulin or fragment thereofcomprises an immunoglobulin heavy chain or light chain variable regioncomprising the amino acid sequence shown in FIG. 8.

In another preferred embodiment, a fully human immunoglobulin orfragment thereof is disclosed, that recognizes at least a portion of ananthrax exotoxin, wherein said immunoglobulin or fragment thereofcomprises an immunoglobulin heavy chain or light chain variable regioncomprising the amino acid sequence shown in FIG. 9.

In another preferred embodiment, a fully human immunoglobulin orfragment thereof is disclosed, that recognizes at least a portion of ananthrax exotoxin, wherein said immunoglobulin or fragment thereofcomprises an immunoglobulin heavy chain or light chain variable regioncomprising the amino acid sequence shown in FIG. 10.

In accordance with other embodiments of the invention the nucleotidesequences shown respectively in FIG. 5 and FIG. 6 are disclosed. Thesenucleotide sequences encode a heavy chain variable region and a lightchain variable region, respectively, of a fully human immunoglobulin orfragment thereof, that recognizes at least a portion of an anthraxexotoxin.

In accordance with other embodiments of the invention, the nucleotidesequences shown in FIG. 8 are disclosed. These nucleotide sequencesencode; a heavy chain variable region and a light chain variable region,respectively, of a fully human immunoglobulin or fragment thereof, thatrecognizes at least a portion of an anthrax exotoxin.

In accordance with other embodiments of the invention, the nucleotidesequences shown in FIG. 9 are disclosed. These nucleotide sequencesencode a heavy chain variable region and a light chain variable region,respectively, of a fully human immunoglobulin or fragment thereof, thatrecognizes at least a portion of an anthrax exotoxin.

In accordance with other embodiments of the invention, the nucleotidesequences shown in FIG. 10 are disclosed. These nucleotide sequencesencode a heavy chain variable region and a light chain variable region,respectively, of a fully human immunoglobulin or fragment thereof, thatrecognizes at least a portion of an anthrax exotoxin.

In one embodiment of the invention, a method for passively immunizing amammal is provided. In one embodiment, the method comprisesadministering an immunizing dose of one or more fully human monoclonalantibodies to a mammal, wherein said one or more fully human monoclonalantibodies comprise an amino acid sequence selected from the groupconsisting of: SEQ ID 2, SEQ ID 4, SEQ ID 6, SEQ ID 8, SEQ ID 10, SEQ ID12, SEQ ID 14, and SEQ ID 16. In one embodiment, the mammal has not beenexposed to anthrax. In another embodiment, the mammal has been exposedto anthrax, and the method is operable to immunize the mammal againstthe effects of subsequent exposures to anthrax.

In one embodiment of the invention, a method for treating a mammal thathas been exposed to anthrax is provided. In one embodiment, the methodcomprises administering a therapeutic dose of one or more fully humanmonoclonal antibodies to a mammal, wherein said one or more fully humanmonoclonal antibodies comprise an amino acid sequence selected from thegroup consisting of: SEQ ID 2, SEQ ID 4, SEQ ID 6, SEQ ID 8, SEQ ID 10,SEQ ID 12, SEQ D 14, and SEQ ID 16.

In one embodiment, the method comprises providing one or moreantibiotics to the mammal. In another embodiment, one or more additionalagents are also given to the mammal, wherein the agent comprises anantibody that contains less than 100% human protein sequences.

In one embodiment, at least two different antibodies are administered toa mammal. In one embodiment, the two antibodies differ by at least oneamino acid. In one embodiment, the two antibodies are administeredsequentially to said mammal (e.g., one antibody immediately after theother, or one antibody within minutes, hours, days, weeks, months etcafter the other). In one embodiment, the two antibodies are administeredsimultaneously to said mammal. In another embodiment, the first antibodybinds to at least one different epitope than the second antibody,thereby exerting a different mechanism of action. In one embodiment,both antibodies work by the same mechanism of action. In someembodiments, three or more antibodies are provided to a mammal. Inpreferred embodiments, one, two or three antibodies are administered forprevention and/or treatment of anthrax. One of skill in the art willunderstand that more than three antibodies can also be administered.

In one embodiment of the invention, the method comprises administeringone or more additional therapeutic agents. Additional therapeutic agentsinclude, but are not limited to, one or more vaccines (e.g., AVA andrPA), antibiotics (e.g., ciprofloxacin hydrochloride, doxycycline, andpenicillin), and/or other antibodies. The additional therapeutic agentscan be administered simultaneously or sequentially with said one or morefully human monoclonal antibodies. In one embodiment, an antibiotic andan antibody are administered shortly after anthrax exposure, followed byadministration of a commercially-available vaccine, such as AVA or rPA.One advantage of such combination therapy is that immediate andlong-term protection can be achieved.

In one embodiment of the present invention, a method of screeninganthrax exotoxin in a sample is provided. In one embodiment, the methodcomprises contacting at least a portion of the sample with one or morefully human monoclonal antibodies comprise an amino acid sequenceselected from the group consisting of: SEQ ID 2, SEQ ID 4, SEQ ID 6, SEQID 8, SEQ ID 10, SEQ ID 12, SEQ ID 14, and SEQ ID 16, and determiningbinding of anthrax exotoxin with said antibody. In one embodiment, thebinding is an indicator of the presence anthrax in said sample, and theabsence of binding is an indicator of the absence of anthrax in saidsample.

In one embodiment of the present invention, a kit to determine thepresence or absence of anthrax exotoxin in a sample is provided. The kitcan be a compilation of materials, an article of manufacture, and/or asystem of materials assembled for a common purpose. In one embodiment,the kit comprises one or more fully human monoclonal antibodiescomprising an amino acid sequence selected from the group consisting of:SEQ ID 2, SEQ ID 4, SEQ ID 6, SEQ ID 8, SEQ ID 10, SEQ ID 12, SEQ ID 14,and SEQ ID 16. Two or more antibodies can be provided in the kit. In afurther embodiment, the kit includes an assay to determine the reactionof anthrax exotoxin with said antibody, wherein said reaction is anindicator of the presence or absence of anthrax in said sample.Instructions regarding the use of the assay can also be included. Theassay can be a binding test. An ELISA can also be used. In oneembodiment, the kit is disposable.

In some embodiments, the kit or method described above is used detectanthrax in biological fluids, such as human serum, saliva, blood cellsetc. In one embodiment, the sample is mammalian tissue. In anotherembodiment, the sample is inorganic or non-biological. In oneembodiment, the kit and method will have utility in determining not justthe presence, but the absence of anthrax contamination.

In one embodiment of the current invention, a kit to protect a mammalfrom anthrax is provided. In one embodiment, the kit comprises one ormore fully human monoclonal antibodies comprises an amino acid sequenceselected from the group consisting of: SEQ ID 2, SEQ ID 4, SEQ ID 6, SEQID 8, SEQ ID 10, SEQ ID 12, SEQ ID 14, and SEQ ID 16. In one embodiment,a medical device for delivering the antibody composition is included.The antibody composition can comprise one antibody, two antibodies, orthree or more antibodies. The composition can be contained within ordisposed onto the medical device. Alternatively, the composition isindependent from said medical device. For example, the kit can include asyringe that contains one or more antibodies in a pre-determined dose.Or, the kit can include a vial of one or more antibodies, which is to bedrawn into the syringe at the time of administration. In one embodiment,the medical device is a syringe, patch, nasal spray, or inhaler.Instructions for using the kit can also be included.

In one embodiment, the kit to protect a mammal from anthrax is to conferimmunity to the mammal, wherein said mammal has not been exposed toanthrax. In another embodiment, the kit is to confer treatment to themammal, wherein the mammal has been exposed to anthrax. In someembodiments, the kit includes an additional therapeutic agent such as anantibiotic (e.g., ciprofloxacin, doxycycline, and/or penicillin). Thekit can also include a vaccine such as AVA and rPA. These additionalingredients can be packaged separately from the monoclonal antibodies,or can be combined with the monoclonal antibodies.

In one embodiment of the present invention, a method for immunizing amammal and/or treating a mammal is provided. In one embodiment, themethod comprises providing an antibody for administration to a mammal.The antibody prevents the assembly of a PA63 heptamer. An effective doseof the antibody is administered to the mammal, thereby preventing theassembly of the PA63 heptamer, thereby inhibiting transport of at leastone of EF and LF into a mammalian host cell, thereby protecting themammal from anthrax infection. Inhibiting the transport of at least oneof EF and LF into the host cell comprises one or more of the followingactions: preventing the entry of said at least one of EF and LF into thehost cell, decreasing the number of said at least one of EF and LF thatenters the host cell, and increasing the length of time for said atleast one of EF and LF to enter the host cell. Inhibiting the transportof EF and/or LF may include any action that disrupts the natural toxiccourse of EF and/or LF.

In one embodiment, the antibody prevents the assembly of the PA63heptamer by binding to a site on a PA83. In another embodiment, themethod comprises providing one or more non-antibody agents that areoperable to inhibit transport of said at least one of EF and LF intosaid mammalian host cell.

In one embodiment, the antibody is a monoclonal antibody. In a furtherembodiment, the antibody is a fully human monoclonal antibody. In yetanother embodiment, the antibody for preventing heptamer assemblycomprises the amino acid sequence selected from the group consisting ofone or more of the following: SEQ ID 2, SEQ ID 4, SEQ ID 14, and SEQ ID16.

In one embodiment, the method for immunizing a mammal and/or treating amammal by preventing PA63 hepatmer formation comprises providing a firstantibody and a second antibody to the mammal, wherein the two antibodiesdiffer by at least one amino acid. In one embodiment, three or moreantibodies are used.

In one embodiment, a pharmaceutical formulation for protecting a mammalfrom one or more toxic effects of anthrax is provided. The formulationcan be for immunizing a mammal or for treating a mammal. In oneembodiment, the formulation includes a binding agent (such as anantibody), wherein the binding agent binds to at least a portion of ananthrax toxin (e.g., PA, EF, and/or LF). In one embodiment, the bindingagent interferes with the assembly of a PA63 oligomer. PA comprises aprotein having a weight of about 83 kD (PA83) that is cleaved into aprotein having a weight of about 63 kD (PA63). The binding agentinhibits the access of at least one of EF and LF to at least a portionof a host mammalian cell, thereby preventing one or more toxic effectsof anthrax in said mammal.

In one embodiment, the pharmaceutical formulation is preventative andformulated for administration to a mammal that has not previously beenexposed to anthrax. In one embodiment, the formulation is to immunize amammal against one or more subsequent exposures to anthrax, and may, insome cases, serve to supplement a preexisting immunity. In anotherembodiment, the pharmaceutical formulation is therapeutic and isformulated for administration to a mammal that has been exposed toanthrax.

In one embodiment, the binding agent is a monoclonal antibody. Inanother embodiment, the binding agent is a fully human monoclonalantibody. In one embodiment, the binding agent comprises the amino acidsequence selected from the group consisting of one or more of thefollowing: SEQ D 2, SEQ ID 4, SEQ ID 6, SEQ ID 8, SEQ ID 10, SEQ ID 12,SEQ ID 14, and SEQ ID 16. In another embodiment, the formulationcomprises one, two or more than two antibodies. In one embodiment, thebinding agent comprises a first binding agent and a second bindingagent, wherein the two binding agents differ by at least one amino acid.The two binding agents may or may not bind to the same portion of theexotoxin. In one embodiment, the two binding agents bind to differentportion of PA, thereby exerting a synergistic effect whenco-administered.

In accordance with another embodiment of the invention, a method isdisclosed for inhibiting the assembly of PA, the binding of the PA toATRS, or the binding of LF or EF to the PA heptamer in a human.Preferably, the method comprises administering to such human theantibody of any of the immunoglobulins or fragments thereof describedabove, including those encoded by the sequences listed in FIGS. 5, 6, 8,9, and 10.

In one embodiment of the present invention, a method of generating afully human monoclonal antibody which recognizes at least a portion ofan anthrax exotoxin is provided. In one embodiment, the method comprisesadministering cells (such as peripheral blood mononuclear cells,lymphocytes) from one or more human donors exposed to anthrax to animmuno-compromised animal, isolating at least one cell from said animal,and fusing the cell(s) with a fusion partner, thereby generating ahybridoma wherein the hybridoma produces a fully human monoclonalantibody which recognizes at least a portion of the anthrax exotoxin. Ina preferred embodiment, one or more fully human monoclonal antibodiesproduced by the methods described herein are provided. In oneembodiment, a cell line that generates fully human antibodies isobtained.

In one embodiment, the method for generating antibodies comprisesscreening the generated antibodies. In another embodiment, the methodincludes transforming at least a portion of the cells with Epstein BarrVirus (EBV). In yet another embodiment, the method comprisescharacterizing the animal's immune response using a test bleed. In afurther embodiment, one or more booster injections of anthrax antigenare administered to the animal. One or more injections of anti-CD8 canalso be administered. In one embodiment, a double selection method(e.g., HAT and ouabain) to select against undesirable cells is used.

In one embodiment, the method for generating antibodies uses cells fromhuman donors that have been vaccinated against anthrax. A human donorthat has been inadvertently exposed to anthrax can also be used.

In one embodiment, the method for generating antibodies usesimmuno-compromised (immuno-deficient) animals such as the SCID mouse.One of skill in the art will understand that the SCID mouse, or othermammal, can be irradiated to further compromise the immune system. Inone embodiment, the fusion partner is a hybridoma. In one embodiment,the fusion partner is a myeloma. In one embodiment, the fusion partneris derived from a mouse myeloma MOPC2 or P3x63Ag8.653.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a timeline of the engraftment of SCID mice with human PBMCfrom anthrax-vaccinated donors.

FIGS. 2A-H show anti-anthrax toxin (PA83) or non-specific IgG levels indonor plasma compared to the engrafted mice.

FIG. 3 shows testing of the presence of neutralizing PA bioactivity indonor and HuPBL-SCID engrafted mice sera.

FIG. 4 shows determination of AVP-21D9 IC50 using RAW 264.7 cell basedassay. AVP-21D9, AVP-22G12 and AVP-1C6 IC50 were assessed at variousconcentrations for the ability to inhibit the lethal toxin.

FIG. 5 shows the nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2)sequence of the 21D9 MAb heavy chain variable region.

FIG. 6 shows the nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4)sequence of the 21D9 MAb light chain variable region.

FIG. 7 shows protection of rats from a lethal toxin challenge.

FIG. 8 shows the nucleotide sequence (SEQ ID NOS: 5 & 7) and amino acidsequence (SEQ ID NOS: 6 & 8) of the 1C6 Mab heavy (SEQ ID NOS: 5 & 6)and light chain (SEQ ID NOS: 7 & 8) variable regions.

FIG. 9 shows the full nucleotide sequence (SEQ ID NOS: 9 & 11) and aminoacid sequence (SEQ ID NOS: 10 & 12) of the 4H7 Mab heavy (SEQ ID NOS: 9& 10) and light chain (SEQ ID NOS: 11 & 12) variable regions.

FIG. 10 shows the full nucleotide sequence (SEQ ID NOS: 13 & 15) andamino acid sequence (SEQ ID NOS: 14 & 16) of the 22G12 Mab heavy (SEQ IDNOS: 13 & 14) and light chain (SEQ ID NOS: 15 & 16) variable regions.

FIG. 11 shows protection of rats from a lethal toxin challenge fiveminutes after administration of antibody.

FIG. 12 shows protection of rats from a lethal toxin challenge byaglycosylated antibody.

FIG. 13 shows protection of rats from a lethal toxin challenge 17 hoursand 1 week after administration of antibody.

FIGS. 14A-B show ELISA panels of AVA vaccinated donors. Volunteersdonors X064-004b and X064-019 plasma obtained at the time of bloodcollection by venipuncture from anthrax-vaccinated donors werepre-screened against tetanus toxoid, PA 83 or LF in an ELISA for bothIgG and IgM.

FIG. 15 shows a sensogram of sequentially bound anti-PA antibodies,demonstrating that irrespective of the order of binding all three humanmonoclonal anti-PA83 antibodies (AVP-21D9, 22G12 and 1C6) can bind to asingle PA83 molecule.

FIGS. 16A-C shows monoclonal antibodies recognizing domains on PA83.FIG. 16A shows a schematic of fragments of PA83 generated by trypsin andchymotrypsin digest based on the sequences and mapping studies. FIG. 16Bshows a Western blot analysis of intact (I), bypsin (T), chymotrypsin(C) and combination of trypsin and chymotrypsin (T+C) generated PAfragments probed with AVP-1C6, AVP-22G12 and AVP-21D9. FIG. 16C showsCoomassie stained SDS-PAGE of antibody bound PA83 treated with trypsinlane (1) Molecular weight markers; (2) PA83 no trypsin; (3) no antibody;(4) AVP-22G12; (5) AVP-21D9; (6) AVP-1C6; (7) AVP-1451 isotype matchedhuman IgG anti-tetanus control.

FIGS. 17A-B show the interaction of human anti-anthrax PA antibodieswith PA63 and lethal factor (FIG. 17A) and PA83 and soluble anthraxtoxin receptor (FIG. 17B) by surface plasmon resonance analysis.

FIG. 18 shows the effects of anti-PA antibodies on PA63 oligomerformation. Coomassie stained SDS-PAGE of antibody bound PA83 treatedwith trypsin. Lane assignment molecular weight markers: (1) PA83 trypsintreated no antibody, (2) AVP-22G12; (3) AVP-21D9; (4) AVP-1C6; (5)AVP-1451 isotype matched human IgG anti-tetanus control.

FIG. 19 shows rat survival data when a combination of AVP-22G12 andAVP-21D9 are administered together.

FIG. 20 shows PA83 detection data in rat serum. Such an assay can beused in one or more kits according to several embodiments of theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A preferred embodiment of the present invention provides a prophylacticor therapeutic agent to counter the effects of anthrax toxin that isreleased as a mechanism of bioterrorism. Thus, in one embodiment of thepresent invention, a potent counter-terrorism measure is provided.Antibodies which bind to one or more components of the tripartiteanthrax exotoxin, the methods of making said antibodies, and the methodsof using said antibodies are provided. In a preferred embodiment, amethod of passive immunization is used to protect a mammal againstanthrax infection. Passive immunization, as used herein, shall be givenits ordinary meaning and shall also mean the introduction of antibodies,for example, from an individual with active immunity, or ofgenetically-engineered or synthetic antibodies, to treat infection.Passive immunization shall also include the administration of one ormore antibodies, or fragments thereof, to confer immunity to a specificpathogen or toxin

In several embodiments, the antibodies provide protection either assingle agents or combined in a cocktail. Anthrax, as defined herein,shall be given its ordinary meaning and shall also include the toxinsecreted by Bacillus anthracis, and shall include the tripartite anthraxtoxin, synthetic or naturally-occurring, and shall also be definedbroadly to include one or more of the following components, synthetic ornaturally-occurring: protective antigen (PA), lethal factor (LF) andedema factor (EF). Thus, antibodies to “anthrax” shall includeantibodies to any portion of one or more components of the anthraxtoxin. Moreover, as used herein, the singular forms “a”, “an”, and “the”include plural reference, unless the context clearly dictates otherwise.Thus, for example, a reference to “a host cell” includes a plurality ofsuch host cells, and a reference to “an antibody” is a reference to oneor more antibodies and equivalents thereof known to those skilled in theart.

The term “fully human antibodies,” as used herein, shall mean antibodieswith 100% human protein sequences. A fully human monoclonal antibody toanthrax may be generated by administering human cells (typically fromone or more human donors exposed to anthrax) to an immuno-compromisedanimal, isolating a lymphocytic cell from that animal, and fusing thelymphocytic cell with a fusion partner, which then produces a fullyhuman monoclonal antibody which recognizes at least a portion of ananthrax exotoxin. The terms “antibody” and “immunoglobulin” shall beused interchangeably. Antibodies for immunizing mammals and/or fortreating mammals are provided in preferred embodiments of the invention.Preferred mammals include humans, but non-human mammals, livestock anddomesticated mammals may also benefit from certain embodiments of theinvention.

As shown generally in FIG. 1, in one embodiment, a method of preparing afully human monoclonal antibody which specifically recognizes at least aportion of the protective antigen (PA) of an anthrax exotoxin isprovided. In one embodiment, this method includes obtaining peripheralblood mononuclear cells from human donors. After obtaining theperipheral blood mononuclear cells from donors, the blood cells areadministered to an immuno-compromised animal. The lymphocytic cells areisolated and fused with a hybridoma fusion partner.

In a preferred embodiment, blood cells from donors who have been exposedto anthrax are obtained. Such exposure may have occurred naturallythrough exposure, or may have occurred by vaccination. Moreover, in oneembodiment, exposure may have occurred decades, years or days prior toobtaining the donor's blood cells. In one embodiment, the “memory” ofsaid exposure is captured or recalled and is selectably expanded byimmunizing the engrafted SCID mice. Thus, in a preferred embodiment,said recall technology is used to generate human monoclonal antibodies.In one embodiment, the human donor has been vaccinated against anthrax.The use of human blood cells that have been “pre-exposed” to anthrax, oranother target antigen, yields surprising and unexpected advantages.These advantages include the generation of antibodies with higheraffinity, higher specificity, and more potent neutralizationcapabilities.

In another embodiment, unexposed or naïve blood cells are used. In oneembodiment, the unexposed blood cells are exposed to anthrax ex vivo orin vitro, prior to engraftment in the immuno-deficient mouse. Thus, saidinitially unexposed cells are transformed into exposed cells and can beused in accordance with the recall technology described above.

In a preferred embodiment, peripheral blood mononuclear cells areobtained from a donor. In another embodiment, other cell types areobtained, including but not limited to lymphocytes, splenocytes, bonemarrow, lymph node cells, and immune cells.

In one embodiment, the blood cells are administered to animmuno-compromised or immuno-deficient animal. In one embodiment, theanimal is a SCID mouse. In one embodiment, the animal is irradiated.

In one aspect of the invention, the animal's immune response ischaracterized using a test bleed. In another embodiment, the generatedantibodies are screened and isolated. In yet another embodiment, thelymphocytic cells are transformed with EBV. In one embodiment, one ormore booster injections of anthrax antigen are administered to theimmuno-compromised animal. In another embodiment, one or more injectionsof anti-human CD8 is administered to the animal. In yet another aspect,a double selection method to select against undesirable cells is used,including, but not limited to using HAT and ouabain. In one embodimentof the present invention, the hybridoma fusion partner is the mousemyeloma P3x63Ag8.653. In another embodiment of the present invention,the hybridoma fusion partner is derived from the mouse myelomaP3x63Ag8.653.

In one embodiment of the present invention, a series of humananti-anthrax PA toxin antibodies is provided. In one embodiment, amonoclonal antibody (AVP-21D9, or “21D9”) is provided. As illustrated inFIG. 4, antibody 21D9 was effective in RAW cell assays in toxicinhibition. The IC50 of 21D9 was found to be in the picomolar range andin approximately equimolar stoichiometry with the input PA toxin. Theequilibrium dissociation constant (K_(d)) as determined by BiaCoreanalysis revealed this embodiment to bind antigen with high affinity inthe picomolar range (Table 1). Deduced amino acid sequence from the 21D9hybridoma heavy and light chain cDNA allowed assignment to known VH andVL gene families, although significant mutation away from these germlinesequences was also observed thereby indicating the occurrence of somatichypermutation. In one embodiment, the mechanism by which 21D9 providesprotection is also provided.

Antibody 21D9, and other antibodies described herein, can be used forhuman use in vivo for prophylaxis and treatment of Anthrax Class Abiowarfare toxins. Thus, in several embodiments, a method for preventinganthrax infection is provided. In one embodiment, a method for treatingmammals to prevent anthrax infection is provided. In a furtherembodiment, a method to treat mammals that have been exposed to anthraxis provided. In particular, FIG. 4 shows the determination of AVP-21D9IC₅₀ using RAW 264.7 cell-based assay. The results demonstrate that 1.2nM PA and 0.56 nM LF in a 96 well assay on confluent RAW 264.7 cellscause 100% cell lysis. The AVP-21D9 was assessed at variousconcentrations for the ability to inhibit the lethal toxin. From thedose response curve an IC₅₀ values was estimated. AVP-1C6 and AVP-22G12IC₅₀ determinations were carried out likewise. The IC₅₀ values forAVP-21D9, AVP-1C6, and AVP-22G12 was 0.21 nM, 0.36 nM, and 0.46 nM,respectively.

In one embodiment, antibody 22G12, and methods of making and using same,are provided. In another embodiment, antibody 1C6, and methods of makingand using same, are provided. In a further embodiment, antibody 4H7, andmethods of making and using same, are provided. Chemical modifications,mutations, and other variants of these antibodies are also provided,including but not limited to 21D9.1 and 22G12.1.

Preferred embodiments provide a fully human monoclonal antibody thatspecifically binds to a component of an anthrax exotoxin or combinationsof components thereof. The anthrax exotoxin can be in tripartite form.The anthrax toxin can be naturally-occurring or synthetic. In atripartite form, the anthrax exotoxin comprises PA, EF, and LF.

In preferred embodiments, a monoclonal antibody is produced by rescuingthe genes encoding antibody variable region from the antibody-producingcells, and establishing stable recombinant cell lines producing wholeIgG/kappa or IgG/lambda. In one embodiment, antibody-producing cellsrecovered from the immunized animal are subjected to cell fusion with anappropriate fusion partner. The resulting hybridomas are then screenedin terms of the activity of the produced antibodies. The hybridomassubjected to selection are screened first in terms of the bindingactivity to a component of the tripartite anthrax exotoxin. In oneembodiment, the hybridomas are selected based on ability to bind to PA,LF and/or EF proteins in an immunoassay, such as an ELISA test and alsoa bioassay. In one embodiment, the hybridoma shows protection in thebioassay. The cells from positive wells are used to isolate mRNA. Fromthe mRNA, cDNA is reverse transcribed. The variable domains are PCRamplified using primers for the 5′ end of the variable chain, andconstant region or frame work primers.

In one embodiment, the amino acid sequences constituting the variableregions of the antibodies having a desired binding activity to PA, LFand/or EF and the nucleotide sequences encoding the same is provided.Several embodiments provide immunoglobulin variable regions containingthe amino acid and nucleotide sequences shown in FIGS. 5,6,8,9, and 10.

FIGS. 5 and 6 show specific heavy chain and light chain variable regionsof 21D9. In one embodiment, one or more of these sequences, or portionsthereof, produce antibodies having a desired binding activity to PA, LFand/or EF. One skilled in the art will appreciate that these sequences,and any products thereof, can comprise one or more variations ormodifications. Variants include amino acid, codon, or base pairsubstitutions, additions, and deletions. In some embodiments, thesechanges are silent such that they do not substantially alter theproperties or activities of the polynucleotide or polypeptide. Variantsalso include alterations in the nucleic acid sequence encoding the aminoacid or peptide sequences. Such variations or modifications may be dueto degeneracy in the genetic code or may be engineered to providedesired properties. Variations or modifications of the nucleotidesequence may or may not result in modifications of the encoded aminoacid sequence.

In a further embodiment, cDNA encoding the immunoglobulin variableregions containing the nucleotide sequences shown in FIG. 5 and FIG. 6,and variants thereof, is provided. In one embodiment, these amino acidsequences or cDNA nucleotide sequences are not necessarily identical butmay vary so long as the specific binding activity to PA, LF and/or EF ismaintained. In another embodiment, variation in nucleotide sequence isaccommodated. In several embodiments, the site corresponding to CDR ishighly variable. In the CDR region, even entire amino acids may vary onsome occasions. Results from experimental data show that in oneembodiment, the heavy chain of 21D9 exhibits a VH3 class, a 3-43 VHlocus, 26 mutations from the germ line, 6-19(1) DH(RF), and JH4B.Results from experimental data show that in one embodiment, the lightchain of 21D9 exhibits a VK1 light chain class, a L12 locus, 14mutations from the germ line, and JK1.

In one embodiment, each immunoglobulin molecule consists of heavy chainshaving a larger molecular weight and light chains having a smallermolecular weight. The heavy and light chains each carries a regioncalled “a variable region” in about 110 amino acid residues at theN-terminus, which are different between the molecules. Variable regionsof a heavy chain and a light chain are designated VH and VL,respectively. The antigen-binding site is formed by forming a dimerbetween the heavy chain variable region VH and the light chain variableregion VL. In one embodiment, the coupling of the antigen-binding siteand the antigen is through electrostatic interaction. The variableregion consists of three CDRs and four frameworks. The CDR forms acomplementary steric structure with the antigen molecule and determinesthe specificity of the antibody. The three CDRs inserted between thefour framework regions (FRs) are present like a mosaic in the variableregion (E. A. Kabat et al., Sequences of proteins of immunologicalinterest, vol. I, 5th edition, NIH Publication, 1991). The amino acidsequences of FRs are well conserved, but those of CDR are highlyvariable and may thus be called hypervariable regions. Among the aminoacid sequences of the antibody specifically recognizing PA, LF and/orEF, a CDR that determines the binding activity to antigens is providedin some embodiments. Preferred embodiments provide CDRs shown in FIG. 5and FIG. 6.

The cDNAs bearing the nucleotide sequences coding the variable regionsin immunoglobulin molecules can be cloned from hybridomas that producethe monoclonal antibody to PA, LF and/or EF of the tripartite anthraxexotoxin. To amplify the sequences, PCR can be performed. To identifyactive clones, ELISA can be used to determine binding to PA, LF and/orEF of the tripartite anthrax exotoxin. Further studies on affinities ofan antibody that can bind to PA, LF and/or EF of the tripartite anthraxexotoxin can be determined with kinetic and thermodynamic studies usingapparatus, such as BiaCore (Biacore, Piscataway, N.J.) surface plasmonresonance apparatus for measuring binding affinity and binding kinetics.Thus, in one embodiment, specific cDNA sequences are provided.

In one embodiment, a monoclonal antibody that can block oligomerizationof the PA component of anthrax exotoxin is provided. Accordingly, amonoclonal antibody of preferred embodiments can have preventive ortherapeutic uses. A preferred monoclonal antibody can be used in apharmaceutical composition as a treatment for a mammal exposed toanthrax exotoxin. Accordingly, preferred embodiments provide methods ofpassive immunization of a mammal against anthrax and/or treating amammal exposed to anthrax.

A monoclonal antibody of several embodiments can be administered as apharmaceutical composition. Thus, in one embodiment, the antibody can beadministered by several different routes, including but not limited to:parenterally, topically, and orally. The term “parenterally”, as usedherein, shall be given its ordinary meaning and shall also includesubcutaneous, intravenous, intraarterial, injection or infusiontechniques, without limitation. In one embodiment, the antibody isadministered intramuscularly. The term “topically”, as used herein,shall be given its ordinary meaning and shall also encompassesadministration rectally and by inhalation spray, as well as the morecommon routes of the skin and the mucous membranes of the mouth andnose. In some embodiments, one or more anti-anthrax antibodies areadministered via a syringe, patch, inhalants, and/or oral formulation.Pre-prepared and pre-dosed anti-anthrax antibody formulations can beavailable in kits so that individuals have easy and quick access to theantibody in the event that those persons are warned of an impendinganthrax exposure or have discovered that they have recently been exposedto anthrax. Such pre-dosed formulations (e.g., syringes, patches,sprays, oral compositions) may be particularly useful for the military.Government workers and individuals working in hospitals may also benefitfrom such anti-anthrax preparations. Such pre-prepared kits may also bemade available to the general public as a safety measure.

One skilled in the art will understand the appropriate dosage to beadministered. Actual dosage levels of preferred antibody in apharmaceutical composition may be varied so as to administer an amountof a preferred antibody that is effective to achieve the desiredtherapeutic response for a particular patient. The selected dosage levelwill depend upon the activity of the particular agent the route ofadministration, the severity of the condition being treated, and thecondition and prior medical history of the patient being treated. Ifdesired, the effective daily dose may be divided into multiple doses forpurposes of administration, e.g., two to four separate doses per day. Itwill be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including thebody weight, general health, diet, time and route of administration,combination with other drugs and the severity of the particular diseasebeing treated.

According to several embodiments of the present invention, thepharmaceutical formulation can be in a variety of forms, including, butnot limited to, injectable fluids, suppositories, powder, tablets,capsules, syrups, suspensions, liquids and elixirs. The preferred routeis by injection. In one embodiment, an antibody preparation ispre-packaged in self-injectable devices, such as syringes. One advantageof such pre-packaged antibody devices is that individuals could protectthemselves on short notice in response to a biological attack, or threatof a biological attack.

Preferred embodiments of the present invention provide a kit foridentifying the presence of anthrax exotoxin in a sample. In a preferredkit, there is a monoclonal antibody which specifically recognizes atleast a portion of a component of an anthrax exotoxin. A sample iscontacted with a monoclonal antibody which specifically recognizes atleast a portion of a component of an anthrax exotoxin. If an anthraxexotoxin is present, then the binding of the anthrax exotoxin with themonoclonal antibody can be determined. The term “kit” as used hereinshall be given its ordinary meaning and shall also include acompilation, collection, or group of materials used for a common goal orpurpose. A kit to test for the presence or absence of anthrax, accordingto on embodiment of the invention includes one or more of the following:an anti-anthrax antibody, a swabbing material, gloves, an assay kit, andinstructions.

In one embodiment, passive immunization is provided in conjunction withone or more other therapies, including but not limited to antibiotictherapy. In one embodiment, ciprofloxacin hydrochloride and/or otherantibiotics are administered before, after, and/or simultaneously withone or more of the antibodies, or fragments thereof, described herein.In some embodiments, the treatment of anthrax infection by two or moretherapies provides a synergistic effect.

The disclosure below is of specific examples setting forth preferredmethods for making agents according to several embodiments of thepresent invention. These examples are not intended to limit the scope,but rather to exemplify preferred embodiments. For example, although thefollowing examples describe the generation of antibodies to anthrax,antibodies to other antigens can also be made by following the examplesset forth below. Various adaptations and modifications to adapt theprotocols described herein will be understood by those skilled in theart.

Example 1 Indirect ELISA

Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated with 50 μlof Bacillus anthracis Protective Antigen (PA) or Lethal Factor (LF)(ListBiological Laboratories (Campbell, Calif.) at a concentration of 1 μg/mLin PBS overnight at 4° C. Plates were washed four times with PBS withTween 20 at 0.1% and 50 μl of diluted sera was added to the wells forone hour at room temperature. Plates were washed as before and 50 μl ofsecondary antibody, HRP conjugated Goat anti-Human IgG, Fcγ specific, orHRP conjugated Goat anti-Human IgM, Fc5μ specific, (JacksonImmunoResearch, West Grove, Pa.) were added and incubated for one hourat room temperature. After another wash step, 100 μL of a substratesolution containing 0.4 mg/mL OPD (O-phenylenediamine dihydrochloride)in citrate buffer (0.025 M at pH 5.0) was added; after 15 minutes, 25 μlof 3N HCl was added to stop the reaction and plates were then read on aMicroplate reader (VersaMax, Molecular Devices, Sunnyvale, Calif.) at490 nm.

Example 2 Raw 264.7 Cell Line In Vitro Bioassay

The presence of neutralizing (protective) antibody to anthrax toxins PAand LF in the antisera were determined using an in vitro protectionbioassay with the mouse macrophage RAW 264.7 target cell line. Hanna, P.et al., Microbiology 90:10198 (1993). PA (100 ng/ml) and LF (50 ng/ml)were pre-incubated with the indicated dilutions of antiserum for 30minutes at 37° C. in a working volume of 100 μl of DMEM mediumsupplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 IU/mlpenicillin and 100 μg/ml streptomycin. This 100 μl volume wassubsequently transferred into a 96 well flat bottom tissue culture platecontaining 1×10⁴ RAW 264.7 cells/well in 100 μl of the same medium. Theculture was incubated for 3 hours at 37° C. The wells were washed twicewith media. The residual attached cells were lysed and the releasedLactate dehydrogenase (LDH) levels were measured using a CytoTox 96 kit(Promega, Madison, Wis.). Briefly, 10 μl Lysis Solution was added to 100μl media per well and the mix incubated 45 minutes in a humidifiedchamber at 37° C., 5% CO₂. An aliquot of the lysed material (50 μl) wastransferred to a new plate and 50 μl assay buffer added. The plate wasincubated for 30 minutes prior to adding 50 μl stop solution. The plateswere read at 490 nm using a Tecan Spectra Fluor (Zurich, Switzerland)reader.

Example 3 Engraftment of SCID Mice with Human PBMC fromAnthrax-Vaccinated Donors

Peripheral blood mononuclear cells were enriched from whole blood ofanthrax-vaccinated donors by density gradient centrifugation usingHistopaque, 1077-1 (Sigma, St. Louis, Mo.). One of skill in the art willunderstand that other types of cells can also be used in accordance withseveral embodiments of the present invention. Typically, one unit ofblood from donors was obtained. Female SCID/bg 12 week old mice wereeach engrafted (via i.p. inoculation) with 2.5×10⁷ isolated human PBMC.They were treated concomitantly i.p. with a volume of conditioned mediumfrom the OKT8 mouse hybridoma grown in Ex-cell 620 hybridoma serum freemedium (JRH, KS) and 2 mM L-glutamine which contained 0.2 mg of theanti-CD8 antibody (used directly without further purification). The micewere immunized with a combination of PA and LF (i.p.) 2 μg each adsorbedto Alum (Imject®, Pierce, Rockford, Ill.) and subsequently boosted(i.p.) on day 7, 19 and day 26. Mice were inoculated with 0.5 ml of EBVobtained from spent conditioned culture medium of the B95-8 marmosetcell line on day 7. Test bleeds were obtained from the orbital sinus ondays 14 and 29. Two consecutive i.p. and iv boosts with PA and LF wereadministered (5 μg via each route on day 40 and 41, both in saline)prior to harvesting cells for fusion on day 42, also at which time anadditional test bleed sample was obtained.

Example 4 Generation of Human Hybridomas

Splenocytes, as well as lymphoblastoid cell line (LCL) tumors wereharvested on day 42 from those mice showing positive test bleeds inindirect ELISA. Human hybridomas were generated from these in separatefusions using a murine myeloma P3x63Ag8.653 with PEG-1500 (Sigma, St.Louis, Mo.) as described by Kearney J F, Radbruch A, Liesegang B,Rajewski K (1979), with the modification that theP3x63Ag8.653:lymphocyte ratio for fusion was between 1:3-1:5. A mousemyeloma cell line that has lost immunoglobulin expression, but permitsthe construction of antibody-secreting hybridoma cell lines. J Immunol123:1548-1558.

Although P3x63Ag8.653 was used in this exemplary method, one skilled inthe art will understand that several fusion partners can be used inaccordance with various embodiments of the current invention, including,but not limited to, cells derived from the mouse myeloma MOPC2, triomas,etc. Double selection to select against the EBV-LCL and the unfusedP3x63Ag8.653 fusion partner was carried out using a combination of HATselection and ouabain. A concentration of 8 μM ouabain (Sigma, St.Louis, Mo.) was used. One skilled in the art will appreciate that otherpoisons or toxins that interfere with the Na+/K+ ATPase can also be usedin accordance with several embodiments of the present invention. Inaddition, one skilled in the art will understand that other selectionmethods can also be used.

Example 5 Treatment and Subcloning of 21D9 Hybridoma Cells

16 days after fusion, hybridoma supernatants from 96 well plates weretested in indirect ELISA. Approximately 17 out of 1248 wells (13 plates)showed an initial positive ELISA signal on PA. All of them were chosenfor further analysis and were subcloned at 5 cells/well on a feederlayer of irradiated NHLF (Cambrex, Baltimore, Md.) in RPMI (Omega, SanDiego, Calif.) supplemented with 10% FBS, 20% hybridoma cloning factor(IGEN, Gaithersbourg, Md.), 5 ng/ml human IL6 (1-188, Leinco), 1× HT(Sigma), 1× Vitamins (Omega), 1× Sodium pyruvate (Omega), 1× NEAA(Omega), 2× L-glutamine (Omega) and without antibiotics. The subcloningplates were tested in indirect ELISA after 10 days. Individual coloniesfrom highly positive wells were hand-picked under a microscope usingPasteur pipets drawn out to fine points. After 2 weeks, individuallypicked clones were retested in indirect ELISA. Positive cells wererecovered and the transcript mRNA encoding the immunoglobulins werereverse transcribed to form cDNA. Although the methodology for antibody21D9 is described herein, one of skill in the art will understand thatthe exemplary methodology described herein can also be used to make andtest the other antibodies described and claimed herein.

Example 6 Variable Region 21D9 IGG and IGK cDNA Cloning and Expression

Total RNA was prepared from specific ELISA positive hybridomas usingRNeasy Mini Kit (Qiagen, Valencia, Calif.). Mixture of VH and VL cDNAswere synthesized and amplified in a same tube using One-Step RT-PCR Kit(Qiagen, Valencia, Calif.). Cycling parameters were 50° C. for 35 min,95° C. for 15 min, 35 cycles of 94° C. for 30 sec, 52° C. for 20 sec and72° C. for 1 min 15 sec, and 72° C. for 5 min.

Primers used for RT-PCR were:

For VHγ Forward a. CVH2 TGCCAGRTCACCTTGARGGAG (SEQ ID NO: 17) b. CVH3TGCSARGTGCAGCTGKTGGAG (SEQ ID NO: 18) c. CVH4 TGCCAGSTGGAGCTRCAGSAG (SEQID NO: 19) d. CVH6 TGCCAGGTACAGCTGCAGCAG (SEQ ID NO: 20) e. CVH1257TGCCAGGTGCAGCTGGTGSARTC (SEQ ID NO: 21) Reverse (located at 5′ of CH1region) a. CγII GCCAGGGGGAAGACSGATG (SEQ ID NO: 22) For VLκ Forward a.VK1F GACATCCRGDTGACCCAGTCTCC (SEQ ID NO: 23) b. VK36FGAAATTGTRWTGACRCAGTCTCC (SEQ ID NO: 24) c. VK2346FGATRTTGTGMTGACBCAGWCTCC (SEQ ID NO: 25) d. VK5F GAAACGACACTCACGCAGTCTC(SEQ ID NO: 26) Reverse (located in constant region) a. Ck543GTTTCTCGTAGTCTGCTTTGCTCA (SEQ ID NO: 27) For VLλ Forward a. VL1CAGTCTGTGYTGACGCAGCCGCC (SEQ ID NO: 28) b. VL2 CAGTCTGYYCTGAYTCAGCCT(SEQ ID NO: 29) c. VL3 TCCTATGAGCTGAYRCAGCYACC (SEQ ID NO: 30) d. VL1459CAGCCTGTGCTGACTCARYC (SEQ ID NO: 31) e. VL78 CAGDCTGTGGTGACYCAGGAGCC(SEQ ID NO: 32) f. VL6 AATTTTATGCTGACTCAGCCCC (SEQ ID NO: 33) Reverse(located in constant region) a. CL2 AGCTCCTCAGAGGAGGGYGG (SEQ ID NO: 34)

The RT-PCR was followed by nested PCR using High Fidelity Platinum PCRMix (Invitrogen, Carlsbad, Calif.). A micro liter of RT-PCR products wasused for VHγ, VLκ or VLλ specific cDNA amplification in the separatetube. At substantially the same time, restriction enzyme sites wereintroduced at both ends. Cycling parameters were 1 cycle of 94° C. for 2min, 6° C. for 30 sec and 68° C. for 45 sec, 35 cycles of 94° C. for 40sec, 54° C. for 25 sec and 68° C. for 45 sec, and 68° C. for 5 min.

Each specific PCR product was separately purified, digested withrestriction enzymes, and subcloned into appropriate mammalianfull-length Ig expression vectors as described below.

Example 7 Subcloning into Vectors

Primers for nested PCR were used. These primers were as follows:

For VH γ Forward (adding BsrGI site at 5′ end) a. BsrGIVHF2AAAATGTACAGTGCCAGRTCACCTTGARGGAG (SEQ ID NO: 35) b. BsrGIVHF3AAAATGTACAGTGCSARGTGCAGCTGKTGGAG (SEQ ID NO: 36) c. BsrGIVHF4AAAATGTACAGTGCCAGSTGCAGCTRCAGSAG (SEQ ID NO: 37) d. BsrGIVHF6AAAATGTACAGTGCCAGGTACAGCTGCAGCAG (SEQ ID NO: 38) e. BsrGIVHF1257AAAATGTACAGTGCCAGGTGCAGCTGGTGSARTC (SEQ ID NO: 39) Reverse (includingnative ApaI Site) a. C γ ER GACSGATGGGCCCTTGGTGGA (SEQ ID NO: 40)

VHγPCR products are digested with BsrG I and Apa I and ligated intopEEG1.1 vector that is linearlized by Spl I and Apa I double digestion.

For VLκ Forward (adding AgeI site, Cys and Asp at 5′end) (SEQ ID NO: 41)a. AgeIVK1F TTTTACCGGTGTGACATCCRGDTGACCCAGTCTCC (SEQ ID NO: 42) b.AgeIVK36F TTTTACCGGTGTGAAATTGTRWTGACRCAGTCTCC (SEQ ID NO: 43) c.AgeIVK2346F TTTTACCGGTGTGATRTTGTGMTGACBCAGWCTCC (SEQ ID NO: 44) d.AgeIVK5F TTTTACCGGTGTGAAACGACACTGACGCAGTCTC Reverse (adding SplI site,located between FR4 and 5′ of constsnt region) (SEQ ID NO: 45) a.SplKFR4R12 TTTCGTACGTTTGAYYTCCASCTTGGTCCCYTG (SEQ ID NO: 46) b.SplKFR4R3 TTTCGTACGTTTSAKATCCACTTTGGTCCCAGG (SEQ ID NO: 47) c. SplKFR4R4TTTCGTACGTTTGATCTCCACCTTGGTCCCTCC (SEQ ID NO: 48) d. SplKFR4R5TTTCGTACGTTTAATCTCCAGTCGTGTCCCTTG

VLκ PCR products are digested with Age I and Spl I and ligated intopEEK1.1 vector linearlized by Xma I and Spl I double digestion.

For VLλ Forward (adding ApaI site at 5′ end) a. ApaIVL1ATATGGGCCCAGTCTGTGYTGACGCAGCCGCC (SEQ ID NO: 49) b. ApaIVL2ATATGGGCCCAGTCTGYYCTGAYTCAGCCT (SEQ ID NO: 50) c. ApaIVL3ATATGGGCCCAGTATGAGCTGAYRCAGCYACC (SEQ ID NO: 51) d. ApaIVL1459ATATGGGCCCAGCCTGTGCTGACTCARYC (SEQ ID NO: 52) e. ApaIVL78ATATGGGCCCAGDCTGTGGTGACYCAGGAGCC (SEQ ID NO: 53) f. ApaIVL6ATATGGGCCCAGTTTTATGCTGACTCAGCCCC (SEQ ID NO: 54) Reverse (adding Avr IIsite, located between FR4 and 5′ of constant region) a. AvrIIVL1IRTTTCCTAGGACGGTGACCTTGGTCCCAGT (SEQ ID NO: 55) b. AvrIIVL237IRTTTCCTAGGACGGTCAGCTTGGTSCCTCCKCCG (SEQ ID NO: 56) c. AvrIIVL6IRTTTCCTAGGACGGTCACCTTGGTGCCACT (SEQ ID NO: 57) d. AvrIIVLmixIRTTTCCTAGGACGGTCARCTKGGTBCCTGC (SEQ ID NO: 58)

VLλPCR products are digested with Apa I and Avr II and ligated intopEELg vector linearlized by Apa I and Avr II double digestion. Thepositive clones were identified after transient co-transfection bydetermining expression in the supernatants by indirect ELISA on PAcoated plates. CHO K1 cells were transfected with different combinationsof IgG and IgK cDNAs using Lipofectamine-2000 (Invitrogen, Carlsbad,Calif.). The supernatants were harvested about 48 hours to about 72hours after transfection. Multiple positive clones were sequenced withthe ABI 3700 automatic sequencer (Applied Biosystems, Foster City,Calif.) and analyzed with Sequencher v4.1.4 software (Gene Codes, AnnArbor, Mich.).

Example 8 Stable Cell Line Establishment

Ig heavy chain or light chain expression vector were double digestedwith Not I and Sal I, and then both fragments were ligated to form adouble gene expression vector. CHO-K1 cells in 6 well-plate weretransfected with the double gene expression vector using Lipofectamine2000 (Invitrogen, Carlsbad, Calif.). After 24 hrs transfection cellswere transferred to a 10 cm dish with selection medium (D.MEMsupplemented with 10% dialyzed FBS, 50 μM L-methionine sulphoximine(MSX), penicillin/streptomycin, GS supplement). Two weeks later MSXresistant transfectants were isolated and expanded. Anti-PA antibodyhigh producing clones were selected by measuring the supernatant with PAspecific ELISA assay. MSX concentration was increased from 50 μM to 100μM to enhance the antibody productivity.

Example 9 Serum Free Adaptation Procedure

A stable cell line was cultured in 10% dialyzed FBS in ExCell 302 SerumFree Medium (JRH, 1000M) with 1×GS (JRH, 100 M) and 25-100 μmL-Methionine Sulphoximine (Sigma). Cells were treated with trypsin(Omega) and split 1:5. The culture medium was switched to 5% FBScontaining media and the cells were cultured 2 days. When the cellsadapted to growing in 5% FBS containing media, the media was changed toserum free medium containing 2.5% dialyzed FBS for 1-2 days, then to100% of serum free media. At this point the cells were no longeradherent and then tranfered to and cultured in Integra flasks for smallscale production in serum free media. Purification was carried out byfiltering the spent culture media through a 0.2μ filter and then loadeddirectly to a HiTrap Protein A column (Pharmacia), followed by washingwith 20 mM Sodium phosphate pH 7.4, and the antibody was eluted with0.1M glycine HCl pH3.4 and immediately neutralized with 1/10 volume of1M Tris-HCl pH 8.0. The protein content in eluted fractions wasdetermined by absorbance at 280 nm, the fractions containing antibodywere pooled and dialyzed against phosphate buffered saline pH 7.4 (2×500volumes), and filter sterilized through a 0.2μ filter. The antibody wasfurther characterized by SDS-PAGE and the purity exceeded 95%.

Example 10 Affinity Determinations

Affinity constants were determined using the principal of surfaceplasmon resonance (SPR) with a Biacore 3000 (Biacore Inc.). A BiacoreCM5 chip was used with affinity purified goat anti-human IgG+A+M(Jackson Immuno Research) conjugated to two flowcells of the CM5 chipaccording to manufacturer's instructions. An optimal concentration of anantibody preparation is first introduced into one of the two flowcells,and is captured by the anti-human Ig. Next, a defined concentration ofantigen is introduced into both flowcells for a defined period of time,using the flowcell without antibody as a reference signal. As antigenbinds to the captured antibody of interest, there is a change in the SPRsignal, which is proportional to the amount of antigen bound. After adefined period of time, antigen solution is replaced with buffer, anddissociation of the antigen from the antibody is then measured, again bythe SPR signal. Curve-fitting software provided by Biacore generatesestimates of the association and dissociation rates, and affinities.

The results from this study are summarized in Table 1, below. Theequilibrium dissociation constant (K_(d)) for recombinant form of the21D9, 1C6, 4H7 and 22G12 MAb was determined by BiaCore analyses. Therate constants k_(on) and k_(off) were evaluated directly from thesensogram in the BiaCore analysis and the K_(d) was deduced.

TABLE 1 Affinity Determination Of Antibody 21D9 And Other Antibodies OnPA (83 Kd) Protein. Dissociation Association Dissociation Constant RateRate Antibody (K_(D)) M (k_(on)) (k_(off)) AVP-21D9 8.21 × 10⁻¹¹ 1.80 ×10⁵ 1.48 × 10⁻⁵ AVP-1C6 7.11 × 10⁻¹⁰ 1.85 × 10⁵ 1.31 × 10⁻⁴ AVP-4H7 1.41× 10⁻¹⁰ 1.74 × 10⁵ 2.45 × 10⁻⁵ AVP-22G12 5.12 × 10⁻¹⁰ 1.01 × 10⁵ 5.17 ×10⁻⁵

Example 11 Human IGG Quantification by Immunoenzymetric Assay

Flat bottom microtiter plates (Nunc F96 Maxisorp) were coated overnightat 4° C. with 50 μl of Goat anti-Human IgG, Fcγ specific, (JacksonImmunoResearch) at 1 μg/mL in PBS. Plates were washed four times withPBS-0.1% Tween 20. Meanwhile, in a separate preparation plate, dilutionsof standards (in duplicate) and unknowns were prepared in 100 μl volumeof PBS with 1 mg/ml BSA. A purified monoclonal human IgG1κ myelomaprotein (Sigma, St. Louis, Mo.) was used as the standard and a differentIgG1κ myeloma protein (Athens Research, Athens, Ga.) served as aninternal calibrator for comparison. Diluted test samples (50 μl) weretransferred to the wells of the assay plate and incubated for one hourat room temperature. Plates were washed as before and 50 μl of thedetecting antibody (1:4000 in PBS with 1 mg/ml BSA.) Goat anti-HumanKappa-HRP (Southern Biotechnology Associates, Inc., Birmingham, Ala.)was added and incubated for one hour at room temperature. After anotherwash step, 100 μl of a substrate solution containing 0.4 mg/ml OPD(O-phenylenediamine dihydrochloride) in citrate buffer (0.025 M at pH5.0) was added. Following a 15 minute substrate incubation, 25 μl of 3NHCl stop solution was added and plates were read on a Microplate reader(VersaMax, Molecular Devices, Sunnyvale, Calif.) at 490 nm. Unknownswere interpolated from standard curve values using SoftMaxPro v4.0software (Sunnyvale, Calif.).

Example 12 Results

Testbleeds from mice engrafted with human PBMC from ananthrax-vaccinated donor and further boosted via immunization in vivowere obtained. FIG. 2A-H shows comparison results of the anti-anthraxtoxin levels in the donor plasma as compared to the sera of engraftedmice. These figures show an IgG response to PA83 in engrafted sera. Thepresence of IgG antibody to anthrax toxin PA83 components in sera ofengrafted SCID mice sera were determined by ELISA after the first andsecond boosts. The specific levels of IgG and donor levels are shown.The IgG response from Donor X064-0042 cells engrafted into SCID mice atday 15 (A) and day 30 (C). The IgG response from Donor X064-043 cellsengrafted into SCID mice at day 15 (E) and day 30 (G). Control data withPBS is also shown.

FIG. 2A-H shows that the mouse sera level of functional immunoreactive(indirect ELISA) antibody is considerably higher than that observed inthe donor. A range of levels of immunoreactive antibody was observed inthe engrafted mice. Test bleeds from engrafted mice were also evaluatedfor the presence of anti-PA/LF protective antibody in the mousemacrophage RAW cell bioassay (FIG. 3). In this bioassay, thetranslocation of PA/LF complex into the cell triggers signaltransduction events (MAPKK mediated) that lead to cell death, and alower bioassay signal. The presence of protective antibody reversesthis. The original donor plasma did not appear to contain detectablelevels of protective antibody (even when tested at a lower dilution) incomparison with the engrafted mice. Both the increase in immunoreactive(ELISA) antibody and the appearance of seroprotection in the engraftedmice show the amplification of a seroprotective immune response toanthrax toxin elicited by repeated immunization of human PBMC-engraftedSCID mice. In one embodiment, the presence of appropriate seropositivityis one preferred criterion for selecting appropriate animals for fusionto generate human hybridomas.

A series of 14 individual fusions was carried out with cells obtainedfrom various compartments (either peritoneal wash (PW), spleen (SP), orLCL tumors (TU) within the peritoneal cavity) of the engrafted mice. Inseveral fusions, the cells were pooled from several engrafted micedetermined to be producing specific anti-anthrax toxin antisera byIndirect ELISA and RAW Cell bioassay prior to fusion. A summary of thefusion results is shown in Table 2.

TABLE 2 Origin Of Resulting Hybridomas Obtained In IJ-8 Anti-AnthraxToxin Study. Cell Positive Plate # Mouse # Sources Partner WellsSubcloned 1 4034/35/37/38/40/41 PW P3X 0 − 2 4034/35/37/38/40/41 PW P3X0 − 3 4034/35/37/38/40/41 PW P3X 0 − 4 4037/38 SP P3X 0 − 5 4034/45 SPP3X 6 + 6 4034/45 SP P3X 10 + 7 4035/40 SP P3X 0 − 8 4035/40 SP P3X 0 −9 4035/40 SP P3X 0 − 10 4035/40 SP P3X 0 − 11 4035/40 SP P3X 1 + 124035/38/40 TU P3X 0 − 13 4035/38/40 TU P3X 0 − 14 4034/45 TU P3X 0 −

Hybridomas were initially selected based on their ability to bind to PA(83 kD) protein adsorbed to polystyrene microtiter plate wells in anindirect ELISA. A wide range of values for the relative amount ofspecific anti-PA antibody in the supernatants was observed. In parallel,each of the supernatants was tested individually in the Anthrax toxinprotection RAW cell bioassay.

A dose-response curve of hybridoma-derived 21D9 in the RAW cell bioassaywas used to evaluate the effective in vitro IC₅₀ protectiveconcentration using a cocktail of the PA (83 kD) and LF toxins. Anantibody IC₅₀ of 0.21 nM was observed for AVP-21D9 and IC50s for theother antibodies are shown in the inset table (FIG. 4).

The 21D9 antibody was found to bind to the intact (83 kD) form as wellas the cleaved (63 kD) form of the PA toxin, but to a lesser degree tothe heptamer as determined by BiaCore analysis (Pharmacia, Peapack,N.J.). Additionally, there was no evidence that the antibody was able toinhibit LF binding to PA (63 kD) heptamer as determined by sequentialincubations in the BiaCore (FIG. 17A). This finding potentiallyimplicates the domain 2 on the PA toxin as the epitope blocked by thisantibody.

The nucleotide sequences of the 21D9 MAb heavy and light chains variableregions were determined (FIG. 5 and FIG. 6).

The alignment of variable regions using V BASE DNAPLOT software (18)showed that 21D9 heavy chain used VH gene from VH3 family (343 locus), Dregion segment 6-19 (in first reading frame) with N region addition andJH4b. 21D9 light chain was from the VKI family (L12 locus), and used theJK1 region segment. The number of mutations from most closely relatedgermline were 26 (heavy chain) and 14 (light chain), respectively.Comparisons with germline V genes suggest that the 21D9 V regions hadundergone extensive somatic mutations, characteristic of an Ag-divenimmune response. Table 3, provided below, shows the germline deviationof the antibodies.

Anthrax exotoxins, the dominant virulence factors produced by Bacillusanthracis are a tripartite combination of protective antigen (PA),lethal factor (LF) and edema factor (EF). Although not wishing to bebound by this theory, these toxins are thought to have an important rolein anthrax pathogenesis; initially to impair the immune system,permitting the anthrax bacterium to evade immune surveillance todisseminate and reach high concentrations; and later in the infectionthe toxins may contribute directly to death in the host animalsincluding humans. Antibodies that neutralize the PA component of theexotoxin could provide an effective protection from anthrax toxinexposure, early and potentially late in the infection. In oneembodiment, the generation of a panel of very potent fully human anti-PAneutralizing antibodies derived from PBMCs obtained from vaccinateddonors is provided.

The antibodies were generated through the combined use of in vivoimmunization of SCID mice reconstituted with human PBMC (U.S. Pat. Nos.5,476,996; 5,698,767; 5,811,524; 5,958,765; 6,413,771; and 6,537,809,all herein incorporated by reference), subsequent recovery of human Bcells expressing anti-PA antibodies and immortalization via cell fusionwith the mouse myeloma cells. Human immunoglobulin cDNAs were isolatedand subcloned into the mammalian expression vector. Recombinantantibodies were first screened by an in vitro neutralization assay usingthe RAW264.7 mouse macrophage cell line. Furthermore, selectedantibodies were evaluated for neutralization of lethal toxin in vivo inthe Fisher 344 rat bolus toxin challenge model (Maynard, 2002; Wild,2003, herein incorporated by reference).

Analysis of the variable regions indicated that antibodies recoveredfrom SCID mice were diverse and hyper-mutated. Among these antibodies, asingle IV administration of AVP-21D9 or AVP-22G12 was found to conferfull protection with only 0.5× (AVP-21D9) or 1× (AVP-22G12) molar excessrelative to PA in the rat toxin challenge prophylaxis model.Aglycosylated PA neutralizing antibodies also protected rats from lethaltoxin challenge. Although not wishing to be bound by the followingtheory, it is believed that the PA toxin neutralizing activity in vivois not depended on Fc mediated effector functions.

In one embodiment, these potent fully human anti-PA toxin-neutralizingantibodies generated may be used for in vivo human use for prophylaxisand/or treatment against Anthrax Class A bioterrorism toxins.

In one embodiment, antibodies that bind to the PA component of thetripartite anthrax-toxin and which provide protection as single agentsare provided. In one embodiment, antibody 21D9 is provided. In anotherembodiment, antibody 22G12 is provided. In a further embodiment,antibody 1C6 is provided. In one embodiment, these antibodies are usedas single agent in preventing and/or treating anthrax infection. Inother embodiments, combination of two or more of these antibodies areused to treat mammals who have been exposed to aerosolized Bacillusanthracis spores, or exposed to other forms of anthrax.

In some embodiments, two or more anti-anthrax antibodies areadministered to the same patient. The antibodies can be administeredsimultaneously, or sequentially. In one embodiment, administration oftwo or more antibodies provided a synergistic effect. For example, FIG.19 shows rat survival data when a combination of AVP-22G12 and AVP-21D9are administered together. When administered together, even at very lowconcentrations, the combination has an enhanced activity as compared tothe individually administered antibodies. It is important to note thatFIG. 19 shows rat survival data in a very sensitive rat model; survivaldata is shown in minutes. Further, although AVP-22G12 is an effectiveanti-anthrax antibody, the survival data for AVP-22G12 in this model issimilar to that for the IgG/K control. This is because extremely lowdoses of AVP-22G12 were administered to the animals. At higher doses,AVP-22G12, like AVP-21D9, is an effective and efficacious anti-anthraxantibody.

In one embodiment, two or more antibodies that exert their actions viadifferent mechanisms are administered to a mammal. In this manner, thetreatment and prevention of anthrax infection may be enhanced becausethe two (or more) antibodies are acting on different pathways. In thismanner, an anti-anthrax embodiment according to one embodiment of theinvention can be combined with an anti-anthrax antibody of the priorart. Further, an anti-anthrax embodiment according to one embodiment ofthe invention can also be combined with another anti-anthraxprophylactic or therapeutic, such an antibiotic.

In one embodiment, antibodies that bind to PA with a range of highaffinities, from about 82 pM to about 700 pM, as determined by surfaceplasmon resonance (BiaCore 3000), is provided. Experimental data showedthat antibodies 1C6, 21D9 and 22G12 recognize unique non-competing sitesand also, 21D9, 22G12 and 1C6 do not appear to interfere with PArecognition of soluble TEM-8. The biological efficacy of these threeantibodies were determined in an in vitro anthrax lethal toxinneutralization assay. All three antibodies protected RAW 264.7 cell fromtoxin induced cell death and provided 50% neutralization atsub-equimolar ratio of antibody to toxin (FIG. 7).

Example 13 Human Monoclonal Antibodies from Anthrax Vaccinated Donorsare Protective Against Anthrax Lethal Toxin In Vivo

A panel of anthrax toxin neutralizing human monoclonal antibodies wasevaluated for neutralization of anthrax lethal toxin in vivo in theFisher 344 rat bolus toxin challenge model. The following experimentcompared five human antibodies that neutralize anthrax lethal toxin invitro in an in vivo rat toxin challenge model. The most potent inhibitorof the anthrax toxin AVP-21D9 protected rat with as little as about 0.5×antibody to toxin in vivo. This corresponds to about 0.12 nmols/200-250g rat. According to one embodiment of the invention, AVP-21D9 was shownto be a potent inhibitor of anthrax toxin in vitro with an estimatedIC₅₀ of 0.2 nM. The potency ranking observed in the in vitro assay wasmatched in the rat in vivo protection assay. Removing the carbohydratesassociated with the constant domains of the IgG did not reduce thepotency of the antibody. In some embodiments, the carbohydrates areuseful for the retention of Fc mediated effector functions. AVP-22G12was also potent at inhibiting the toxin in vivo at 1×, but not as potentas AVP-21D9 at the 0.5× dose. Removal of the glycosylation site inAVP-22G12 did impact on its potency suggesting that although theeffector functions are not required, in the absence of the carbohydratesthe overall structure of the antibody is impacted to reduce its efficacyto 80% survival at the designated 5 hour time point, which dropped to60% due to an additional death at 12 hours. At the lower dose ofAVP-22G12 no protection was observed but the time to death was delayedsignificantly. AVP-1C6 at 1× was only 80% protective and failed toprotect or delay time to death at the lower dose. The in vivo potencytrend observed AVP21D9>AVP-22G12>AVP-1C6, is the similar to the potencyin vitro and correlates well with affinity of antibody to PA.

Accordingly, vaccination with Anthrax Vaccine Adsorbed can induce theproduction of a range of protective antibodies. The experiments showedthat the human anti-anthrax toxin antibodies according to severalembodiments of the invention are potent inhibitors of the lethal toxinin vivo. The three parental antibodies and the two aglycosylated formsdescribed may be therapeutically useful against anthrax infection and inthe passive protection of high risk individuals. In particular, the twomost potent anthrax toxin-neutralizing antibody (AVP-21D9 and AVP-22G12)were completely effective at a dose corresponding to 0.12 nmols/rat and0.25 nmols/rat respectively. One of skill in the art will understand theappropriate dosages to prevent or treat anthrax infection in humans andother animals.

Detailed methodology and results are described below.

Methods

The in vivo anthrax toxin neutralization experiments were performedbasically as described by Ivins B E, Ristroph J D, Nelson G O: Influenceof body weight on response of Fischer 344 rats to anthrax lethal toxin.Appl Environ Microbiol, 1989, 55(8):2098, herein incorporated byreference. Male Fisher 344 rats with jugular vein catheters weighingbetween 200-250 g were purchased from Charles River Laboratories(Wilmington, Mass.). Human anti-anthrax PA IgG monoclonal antibodiesAVP-21D9, AVP-22G12, AVP-1C6, AVP-21D9.1 and AVP22G12.1 were producedfrom recombinant CHO cell lines adapted for growth in serum free media.The human IgG monoclonal antibodies were purified by affinitychromatography on HiTrap Protein A, dialysed against PBS pH7.4 andfilter sterilized. Rats were anaesthetized in an Isofluorane (Abbot, II)EZ-anesthesia chamber (Euthanex Corp., PA) following manufacturesguidelines. The antibody was administered via the catheter in 0.2 mlPBS/0.1% BSA (pH 7.4) and at either 5 minutes, 17 hour or a week laterlethal toxin (PA 20 μg/LF 4 μg in 0.2 ml PBS/0.1% BSA (pH 7.4)) wasadministered via the same route. Five animals were used in each testgroup and four animals in each control. Test and control experimentswere carried out at the same time using the same batch of reconstitutedPA and LF toxins (List Laboratories, CA). Animals were monitored fordiscomfort and time of death versus survival, as assessed on the basisof cessation of breathing and heartbeat. Rats were maintained underanaesthesia for 5 hr post exposure to lethal toxin or until death tominimize discomfort. Rats that survived were monitored for 24 hours andthen euthanized by carbon dioxide asphyxiation.

Results

Effect Of Anti-Anthrax PA Antibodies On Protection Of Rats From LethalToxin Challenge: FIG. 11 illustrates the protection profile of the threeantibodies AVP-21D9, 22G12 and 1C6 in the rat model at two doses 0.5×and 1+ molar ratios relative to toxin challenge. AVP-21D9 protected ratsat 0.5× and no deaths were observed in the 5 hr following toxinadministration, likewise AVP-22G12 at 1× also showed completeprotection. However with AVP22G12 at 0.5× the time to death wasprolonged to 255 min. The administrations of lethal toxin 5 min afterthe infusion of 0.5× or 1× control human control IgG resulted in time todeath of 85-120 min. AVP-1C6 at 1× conferred 80% protection and at 0.5×was not protective.

Effect of Antibody Glycosylation on Anti-Anthrax PA Antibodies onProtection of Rats from Lethal Toxin Challenge: Aglycosylated antibodiescorresponding to AVP-21 D9 or AVP-22G12 were generated by mutating aN-glycosylation site (N297Q) in the Fc region. These antibodies aredesignated as AVP-21D9.1 and AVP-22G12.1, respectively and compared tothe glycosylated counterparts in the rat toxin challenge prophylaxismodel. As described above, antibody was intravenously administered 5minutes prior to the lethal toxin (PA/LF) challenge. Both AVP-21D9 andAVP-21D9.1 fully protected rats against anthrax toxin with 0.5× molarexcess relative to PA toxin, whilst AVP-22G12.1 was slightly less potentthan the parent molecule at 1× as shown in FIG. 12.

Duration of AVP-21D9 Antibody Mediated Protection of Rats from LethalToxin Challenge: To investigate the duration of protection afforded by afully human antibody in Fischer rats AVP-21D9 was intravenouslyadministered 17 hours or 1 week prior to the lethal toxin (PA/LF)challenge. As shown in FIG. 13, a single administration of AVP-21D9 at1× protected 100% when challenged 17 hrs later. Over the extended periodof time administration of AVP-21D9 at 10× dose showed 80% protection.Almost all control animals died within 120 min, one outlier had delayedtime of death to 230 min.

Accordingly, the results showed that a single IV dose of AVP-21D9 orAVP-22G12 was found to confer full protection with only 0.5× (AVP-21D9)and 1× (AVP-22G12) molar excess relative to the anthrax toxin in the ratchallenge prophylaxis model. AVP-21D9, AVP-22G12 and AVP-1C6 protectrats from anthrax lethal toxin at low dose. Aglycosylated versions ofthe most potent antibodies are also protective in vivo in the rat model,according to one embodiment. The protective effect of AVP-21D9 persistsfor at least one week in rats. These potent fully human anti-PAtoxin-neutralizing antibodies are attractive candidates for developmentfor in vivo human use as prophylaxis and/or treatment against AnthraxClass A bioterrorism toxins.

Example 14 Human Anti-Anthrax Protective Antigen Neutralizing MonoclonalAntibodies Derived from Donors Vaccinated with Anthrax Vaccine Adsorbed

Potent anthrax toxin neutralizing human monoclonal antibodies weregenerated from peripheral blood lymphocytes obtained from AnthraxVaccine Adsorbed (AVA) immune donors. In this particular experiment,donors were recruited that had been actively immunized with the currentlicensed anthrax vaccine (AVA). Despite vaccination the serum levels ofanti-PA83 specific IgG and IgM were relatively low (2-3 μg/ml) incomparison to the anti-tetanus responses in both donors. In thisembodiment, a SCID-HuPBL platform was used to demonstrate that theinventors could selectively direct the recall response by immunizationof the chimeric animals. Immunization of the chimeric mice withrecombinant PA83 resulted in a significant increase in specific IgG insome of the engrafted mice, in one case as high as 2 mg/ml (mouse 4152).In comparing first and second bleeds for both sets of chimeric mice, itis clear that a specific response was selectively enhanced in theanimals upon boosting with antigen (see FIG. 2).

Not wishing to be bound by the following theory, it is believed that inmice that responded well to antigen challenge, the inventors recalledthe human memory B cell response and recruited specific human helperT-cells. The specific recall leads to proliferation of antigen specificplasma cells.

The antibody producing cells in the chimeric mice were recovered fromthe spleen and peritoneal washes in sufficient numbers to permit fusionwith a standard mouse myeloma P3X63Ag8.653 (Kearney J F, Radbruch A,Liesegang B, Rajewsky K: A new mouse myeloma cell line that has lostimmunoglobulin expression but permits the construction ofantibody-secreting hybrid cell lines. J Immunol, 1979. 123(4):1548,herein incorporated by reference) to form hybridomas. The formation ofmouse/human hybridomas using a murine fusion partner with human derivedplasma cells can result in unstable hybrids, which can be challenging toclone, expand and isolate. Accordingly, in one embodiment, the inventorsrescued the transcripts encoded by mRNA from a small cluster of cellsand generating stable recombinant CHO cell lines and testing these forthe activity. Hence the fusion with P3X63Ag8.653 with the human cellsresults in hybrids of antibody-producing cell, which permitsidentification of positive wells for specific IgG production and therescue of immunoglobulin transcripts.

No particular heavy chain family or light chains dominated the humananti-PA response. In all but two cases, the inventors could assign DHsegments usage. The array of J_(H) and J_(L) segments observed in thepanel suggest that the approach is capturing the diversity present inthe natural response to anthrax PA via vaccination with AVA. Anotherstriking feature of the antibodies, according to one embodiment, is theexceptional high affinity for the target antigen and the very slowoff-rates. Similar high affinities and slow off rates for anti-tetanustoxoid antibodies derived from engrafted HuPBL-SCID mice boosted withantigen. Thus, this may be a general feature of the protectiveanti-bacterial toxin response in humans.

Currently in the event of an inadvertent Bacillus anthracis sporeexposure, two preventative measures can be taken. If the risk can beassessed well in advance, vaccination can be employed. In the event ofnear term or immediate post exposure antibiotic such as Cipro may beeffective. Anthrax Vaccine Adsorbed (AVA) is the only licensed humananthrax vaccine in the United States. The vaccine is known to contain amixture of cell products including PA, LF and EF, however the exactamounts are unknown (Turnbull P C, Broster M G, Carman J A, Manchee R J,Melling J: Development of antibodies to protective antigen and lethalfactor components of anthrax toxin in humans and guinea pigs and theirrelevance to protective immunity. Infect Immun, 1986. 52(2):356, hereinincorporated by reference). The immunization schedule consists of threesubcutaneous injections at 0, 2 and 4 weeks and booster vaccination at6, 12 and 18 months and it is suggested that annual boost may berequired to maintain immunity. Mass vaccination in the event of anthraxspore release is an unlikely scenario. First, the time taken foreffectiveness of such vaccination based on AVA or various rPA moleculesin development may be too short, weeks as opposed to minutes. Theutilization of antibiotic can inhibit bacterial growth and spread andmay prevent some of the symptoms, but the administration needs to betimely and preferably prophylactically, even as such, the toxinsreleased during the early stages of an infection may impair the immunesystem to cause lasting damage. In some instances, a combination ofinhibiting anthrax bacteria and toxins is required early in aninfection. High affinity human monoclonal PA neutralizing antibodies mayprovide immediate neutralization of the anthrax toxins.

In this experiment, the inventors accessed the human IgG response to thePA83 component of AVA and isolated a panel of high affinity potent PAneutralizing monoclonal antibodies. These antibodies were selected onthe basis of binding to PA83, the form of the anthrax toxin released bythe bacteria prior to cell bound furin processing and lethal toxininhibition. Some of the embodiments described herein will beparticularly useful for the generation of fully human monoclonalantibodies against various infectious disease targets from vaccinated ornaturally exposed yet protected individuals.

The specific methodology and results of the experiment are described indetail as follows.

Methods

Selection of Donor: Plasma obtained at the time of blood collection byvenipuncture from anthrax-vaccinated donors were pre-screened against apanel of antigens (including components of the anthrax exotoxin PA andLF) in an ELISA for both IgG and IgM. An internal calibrator wasincorporated into each assay consisting of a control antiserumcontaining both IgG and IgM anti-tetanus toxoid. The IgG and IgM titreswere compared across assays performed on different days, therebypermitting more robust comparisons of the entire donor panel.

Engraftment of SCID mice with human PBMC from pre-selected AVA immunedonors: Peripheral blood mononuclear cells were enriched from wholeblood of AVA immune donors by density gradient using Histopaque. SCID/bg12 week old mice were each engrafted (via i.p. injection) with 2.5×10⁷human PBMC. They were treated concomitantly i.p. with a volume ofconditioned medium which contains 0.2 mg of the anti-CD8 monoclonalantibody. The mice were immediately immunized (i.p.) with therecombinant PA and LF (List Laboratories) 10 μg each adsorbed to Alum(Imject®, Pierce, Rockford, Ill.) and subsequently boosted (ip) 8-28 daylater. Mice were inoculated with 0.5 ml of EBV obtained from spentconditioned culture medium day 15 following engraftment. Test bleedswere obtained from the orbital sinus, on days 15 and 30. Two consecutiveiv and i.p. boosts with the appropriate toxins were administered(typically, 5 μg each on day 35 and day 36; both in saline) prior toharvesting cells for fusion on day 37, also at which time an additionaltest bleed sample was obtained. The total IgG and specific PA IgGcombined with potency in the RAW 264.7 cell bioassay were determined forthe bleeds.

Generation of human hybridomas: Splenocytes, peritoneal washes, as wellas lymphoblastoid cell line (LCL) human lymphocyte derived tumors, wereharvested on day 37 from those mice showing positive test bleeds in PAELISA and the appropriate bioassay (described below). Human hybridomaswere generated from these in separate fusions using a mouse myeloma cellline P3X/63Ag8.653 (Kearney J F, Radbruch A, Liesegang B, Rajewsky K: Anew mouse myeloma cell line that has lost immunoglobulin expression butpermits the construction of antibody-secreting hybrid cell lines. JImmunol, 1979, 123(4):1548, herein incorporated by reference) withPEG-1500. Double selection to select against the EBV-LCL and theun-fused fusion partner was carried out using a combination of HATselection and ouabain.

Variable Region IgH and IgL cDNA cloning and expression: Total RNA wasprepared from specific ELISA positive hybridomas using RNeasy Mini Kit(Qiagen, Valencia, Calif.). Mixture of VH and VL cDNAs were synthesizedand amplified in a same tube using One-Step RT-PCR Kit (Qiagen,Valencia, Calif.). Cycling parameters were 50° C. for 35 min, 95° C. for15 min, 35 cycles of 94° C. for 30 sec, 52° C. for 20 sec and 72° C. for1 min 15 sec, and 72° C. for 5 min.

Primers used for RT-PCR were:

For VHγ Forward a. CVH2 TGCCAGRTCACCTTGARGGAG (SEQ ID NO: 17) b. CVH3TGCSARGTGCAGCTGKTGGAG (SEQ ID NO: 18) c. CVH4 TGCCAGSTGCAGCTRCAGSAG (SEQID NO: 19) d. CVH6 TGCCAGGTACAGCTGCAGCAG (SEQ ID NO: 20) e. CVH1257TGCCAGGTGCAGCTGGTGSARTC (SEQ ID NO: 21) Reverse (located at 5′ of CH1region) a. CγII GCCAGGGGGAAGACSGATG (SEQ ID NO: 22) For VLκ Forward a.VK1F GACATCCRGDTGACCCAGTCTCC (SEQ ID NO: 23) b. VK36FGAAATTGTRWTGACRCAGTCTCC (SEQ ID NO: 24) c. VK2346FGATRTTGTGMTGACBCAGWCTCC (SEQ ID NO: 25) d. VK5F GAAACGACACTCACGCAGTCTC(SEQ ID NO: 26) Reverse (located in constant region) a. Ck543GTTTCTCGTAGTCTGCTTTGCTCA (SEQ ID NO: 27) For VLλ Forward a. VL1CAGTCTGTGYTGACGCAGCCGCC (SEQ ID NO: 28) b. VL2 CAGTCTGYYCTGAYTCAGCCT(SEQ ID NO: 29) c. VL3 TCCTATGAGCTGAYRCAGCYACC (SEQ ID NO: 30) d. VL1459CAGCCTGTGCTGACTCARYC (SEQ ID NO: 31) e. VL78 CAGDCTGTGGTGACYCAGGAGCC(SEQ ID NO: 32) f. VL6 AATTTTATGCTGACTCAGCCCC (SEQ ID NO: 33) Reverse(located in constant region) a. CL2 AGCTCCTCAGAGGAGGGYGG (SEQ ID NO: 34)

The RT-PCR was followed by nested PCR with High Fidelity Platinum PCRMix (Invitrogen, Carlsbad, Calif.). A microliter of RT-PCR products wereused for VHγ, VLκ or VLλ specific cDNA amplification in the separatetube. At the same time restriction enzyme sites were introduced at bothends. Cycling parameters were 1 cycle of 94° C. for 2 min, 60° C. for 30sec and 68° C. for 45 sec, 35 cycles of 94° C. for 40 sec, 54° C. for 25sec and 68° C. for 45 sec, and 68° C. for 5 min.

The each specific PCR products were separately purified, digested withrestriction enzymes, and subcloned into appropriate mammalianfull-length Ig expression vectors as described below.

Primers for nested PCR were: For VH γ Forward (adding BsrGI site at5′ end) a. BsrGIVHF2 AAAATGTACAGTGCCAGRTCACCTTGARGGAG (SEQ ID NO: 35) b.BsrGIVHF3 AAAATGTACAGTGCSARGTGCAGCTGKTGGAG (SEQ ID NO: 36) c. BsrGIVHF4AAAATGTACAGTGCCAGSTGCAGCTRCAGSAG (SEQ ID NO: 37) d. BsrGIVHF6AAAATGTACAGTGCCAGGTACAGCTGCAGCAG (SEQ ID NO: 38) e. BsrGIVHF1257AAAATGTACAGTGCCAGGTGCAGCTGGTGSARTC (SEQ ID NO: 39) Reverse (includingnative ApaI site) a. C γ ER GACSGATGGGCCCTTGGTGGA (SEQ ID NO: 40)

VHγPCR products are digested with BsrG I and Apa I and ligated intopEEG1.1 vector that is linearlized by Spl I and Apa, I double digestion.

For VLκ Forward (adding AgeI site, Cys and Asp at 5′end) (SEQ ID NO: 41)a. AgeIVK1F TTTTACCGGTGTGACATCCRGDTGACCCAGTCTCC (SEQ ID NO: 42) b.AgeIVK36F TTTTACCGGTGTGAAATTGTRWTGACRCAGTCTCC (SEQ ID NO: 43) c.AgeIVK2346F TTTTACCGGTGTGATRTTGTGMTGACBCAGWGTCC (SEQ ID NO: 44) d.AgeIVK5F TTTTACCGGTGTGAAACGACACTCACGCAGTCTC Reverse (adding SplI site,located between FR4 and 5′ of constant region) (SEQ ID NO: 45) a.SplKFR4R12 TTTCGTACGTTTGAYYTCCASCTTGGTCCCYTG (SEQ ID NO: 46) b.SplKFR4R3 TTTCGTACGTTTSAKATCCACTTTGGTCCCAGG (SEQ ID NO: 47) c. SplKFR4R4TTTCGTACGTTTGATCTCCACCTTGGTCCCTCC (SEQ ID NO: 48) d. SplKFR4R5TTTCGTACGTTTAATCTCCAGTCGTGTCCCTTG

VLκ PCR products were digested with Age I and Spl I and ligated intopEEK1.1 vector linearlized by Xma I and Spl I double digestion.

For VLλ Forward (adding ApaI site at 5′end) a. ApaIVL1ATATGGGCCCAGTCTGTGYTGACGCAGCCGCC (SEQ ID NO: 49) b. ApaIVL2ATATGGGCCCAGTCTGYYCTGAYTCAGCCT (SEQ ID NO: 50) c. ApaIVL3ATATGGGCCCAGTATGAGCTGAYRCAGCYACC (SEQ ID NO: 51) d. ApaIVL1459ATATGGGCCCAGCCTGTGCTGACTCARYC (SEQ ID NO: 52) e. ApaIVL78ATATGGGCCCAGDCTGTGGTGACYCAGGAGCC (SEQ ID NO: 53) f. ApaIVL6ATATGGGCCCAGTTTTATGCTGACTGAGCCCC (SEQ ID NO: 54) Reverse (adding Avr IIsite, located between FR4 and 5′ of constant region) a. AvrIIVL1IRTTTCCTAGGACGGTGACCTTGGTCCCAGT (SEQ ID NO: 55) b. AvrIIVL237IRTTTCCTAGGACGGTCAGCTTGGTSCCTCCKCCG (SEQ ID NO: 56) c. AvrIIVL6IRTTTCCTAGGACGGTCACCTTGGTGCCACT (SEQ ID NO: 57) d. AvrIIVLmixIRTTTCCTAGGACGGTCARCTKGGTBCCTCC (SEQ ID NO: 58)

VLλ PCR products were digested with Apa I and Avr II and ligated intopEELg vector linearlized by Apa I and Avr II double digestion.

The positive clones were identified after transient co-transfection bydetermining expression in the supernatants by indirect ELISA on PAcoated plates. CHO K1 cells were transfected with different combinationsof IgG and IgK cDNAs using Lipofectamine-2000 (Invitrogen, Carlsbad,Calif.). The supernatants were harvested 48-72 h after transfection.Multiple positive clones were sequenced with the ABI 3700 automaticsequencer (Applied Biosystems, Foster City, Calif.) and analyzed withSequencher v4.1.4 software (Gene Codes, Ann Arbor, Mich.).

Stable cell line establishment: Ig heavy chain or light chain expressionvector were double digested with Not I and Sal I, and then bothfragments were ligated to form a double gene expression vector. CHO-K1cells in 6 well-plate were transfected with the double gene expressionvector using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). After 24hrs transfection cells were transferred to 10 cm dish with selectionmedium (D.MEM supplemented with 10% dialyzed FBS, 50 μM L-methioninesulphoximine (MSX), penicillin/streptomycin, GS supplement). Two weekslater MSX resistant transfectants were isolated and expanded. Highproducing clones were selected by measuring the antibody levels insupernatants in a PA specific ELISA assay. MSX concentration wasincreased from 50 to 100 μM to enhance the antibody productivity.

Antigen Binding ELISA: The presence of antibody to anthrax toxincomponents in human sera, engrafted SCID mouse sera, supernatants ofhybridomas or transiently transfected CHO-K1 cells were determined byELISA. Briefly flat bottom microtiter plates (Nunc F96 Maxisorp) werecoated with the appropriate component of the Bacillus anthracistripartite exotoxin, such as PA or LF, diluted sera was added to thewells for one hour at room temperature. Plates were washed and secondaryantibody, goat anti-human IgG:HRP, Fcγ specific, or goat anti-humanIgM:HRP, Fcμ specific was added and incubated for one hour at roomtemperature. After another wash step, a substrate solution containingOPD (O-phenylenediamine dihydrochloride) in citrate buffer was added.After 15 minutes, 3N HCl was added to stop the reaction and plates wereread on a Microplate reader at 490 nm.

Human Ig/κ/λ Quantification by ELISA: Flat bottom microtiter plates(Nunc F96 Maxisorp) were coated overnight at 4° C. with 50 μl of goatanti-human IgG, Fcγ specific, at 1 μg/ml in PBS. Plates were washed fourtimes with PBS-0.1% Tween 20. Meanwhile, in a separate preparationplate: dilutions of standards (in duplicate) and unknowns were preparedin 100 μl volume of PBS with 1 mg/ml BSA. A purified monoclonal humanIgG1/κ or λ protein was used as the standard and a different IgG1/κ or λprotein serves as an internal calibrator for comparison. Diluted testsamples (50 μl) were transferred to the wells of the assay plate andincubated for one hour at room temperature. Plates were washed as beforeand 50 μl of the detecting antibody, goat anti-human kappa or lambda-HRPwas added, incubated for one hour at room temperature, and developed asdescribed above. RAW 264.7 cell line in vitro bioassays were performedas described earlier.

Binding Affinity Determinations: Affinity constants were determinedusing the principal of surface plasmon resonance (SPR) with a BiaCore3000 (BiaCore Inc.). Affinity purified goat anti-human IgG (JacksonImmunoResearch) was conjugated to two flow cells of the CM5 chipaccording to manufacturer's instructions. An optimal concentration of anantibody preparation was first introduced into one of the two flowcells,and was captured by the anti-human IgG. Next, a defined concentration ofantigen was introduced into both flow cells for a defined period oftime, using the flow cell without antibody as a reference signal. Asantigen bound to the captured antibody of interest, there was a changein the SPR signal, which was proportional to the amount of antigenbound. After a defined period of time, antigen solution was replacedwith buffer, and the dissociation of the antigen from the antibody wasthen measured, again by the SPR signal. Curve-fitting software providedby BiaCore generated estimates of the association and dissociation ratesfrom which affinities were calculated.

Results

General: The range of antibodies generated were diverse with evidence ofextensive hyper mutation, and all were of very high affinity forPA83˜1×10⁻¹⁰⁻¹¹M. Moreover, all were potent inhibitor of anthrax lethaltoxin in vitro. Accordingly, in one embodiment, the generation of apanel of potent human monoclonal antibodies derived from anthrax vaccineadsorbed immune donors is provided. Protection against anthrax toxinchallenge in an in vitro cell culture assay correlates well withaffinity, with the highest affinity antibody AVP-21D9 (Kd=82 μM)exhibiting the most potent toxin inhibition.

Donor Screening: Donors sera (X064-004b and X064-019) were screened forIgG and IgM against tetanus toxoid, PA and LF by ELISA. FIG. 14 showsthat both donors had significant IgG responses to tetanus toxoid andsome albeit low levels of specific IgG antibody against PA and LF.

Chimeric Engraft Screening: The PBL's from donor X064-004b and X064-019were engrafted into mice designated X040-042 and X040-043 respectively.After boosting, sera from engrafted mice were screened for human IgGagainst PA. As shown in FIG. 2, the initial bleed after the first boostis plotted alongside the X064-004b donor sera. One engraft had ananti-PA IgG level that is 9× higher than the donor sera. Moreover, asshown in FIG. 2, the second bleed from the engrafted mice, a range of8-30 fold increase in specific anti-PA IgG is observed. This increase inspecific IgG over time in the engrafted mice is even more pronounced inthe second engraft using cells from donor X064-01. The increase inspecific anti-PA IgG in the second bleed is more than 500 fold relativeto specific anti-PA in the donor sera.

Immunoglobulin Sequence Analysis: Following fusion, cells producinghuman anti-anthrax PA IgG were selected and the cDNA encoding theimmunoglobulin variable regions were rescued from ELISA positive wellsand sequenced. The cDNA templates were used to establish stable CHO K1cell lines producing antibodies. Four neutralizing anti-PA antibodieswere discovered by this approach. The VH families were represented bythe VH3, VH1 and VH4. Likewise the VL families were represented by VK Iand VL 3. Both VH and VL regions contained evidence of hyper mutationaway from the germline. Table 3, below, lists the antibodies isolated bythis approach and the D and J regions are assigned where possible usingDNAPLOT from Vbase.

TABLE 3 Human Anti-Anthrax PA83 Antibody Classification VH VL #Mutations # Mutations from from Designation VH Class VH Locus germlineDH(RF) JH VL Class VL Locus Germline JL AVP-21D9 VH3 3-43 26 6-19(1)JH4b VKI L12 14 JK1 AVP-1O6 VH3 3-73 8 6-13(1) JH3b/a VKI L18 13 JK4AVP-4H7 VH4 4-39 29 unknown JH6b/a VL3 3 h 22 JL2/JL3a AVP-22G12 VH33-11 20 unknown JH5b VL3 3r 9 JL2/JL3a

The immunoglobulin sequence derived from the cDNA encoding the variableregions were used to search Vbase and the VH class, VH locus, DH and JHsegments were assigned for the VH regions. Likewise VL class, VL locusand JL segments were assigned for the VL regions. Comparing the actualsequences and closest matched V family members the extent of somatichyper mutation could be ascertained.

Kinetics of Binding: The equilibrium dissociation constant (K_(d)) forrecombinant form of the antibodies were determined by BiaCore analyses.The rate constants k_(on) and k_(off) were evaluated directly from thesensogram in the BiaCore analysis and the K_(d) was deduced. The resultsare summarized in Table 1, provided above.

In one embodiment, one feature of many of the protective antibodies isthe very slow off rate, which contribute to the very high affinities8.21×10⁻¹¹ M to 7.11×10⁻¹⁰M. The slow off rate may confer significantphysiological advantages for toxin neutralization in vivo.

In vitro Lethal Toxin Inhibition: All the antibodies were initiallyselected based on binding to PA83 and secondly on inhibition of lethaltoxin in a Raw 264.7 cell based in vitro assay. Only clones exhibitingtoxin neutralization in a qualitative assay were developed further. TheRaw 264.7 cell assay was adapted to compare the various antibodies forpotency of toxin inhibition. In FIG. 4 a typical antibody dose responsecurve is reconstructed to provide an estimate of the IC₅₀ for AVP-21D9,AVP-1C6 and AVP-22G12. Again, the inhibitory potency ranking of all theselected antibodies were reflecting the same ranking observed for thebinding to PA83.

Example 15 Synergistic Effects of Anti-Anthrax Antibody andCiprofloxacin

In one embodiment of the invention, passive immunization is provided inconjunction with one or more other therapies, including but not limitedto antibiotic therapy. In one embodiment, ciprofloxacin hydrochlorideand/or other antibiotics are administered before, after, and/orsimultaneously with one or more of the antibodies, or fragments thereof,described herein. In one embodiment, one or more antibiotics andantibodies are administered first, followed by vaccination with AVAand/or rPA, thereby conferring both immediate and long-term protection.In other embodiments, doxycycline can be administered. In otherembodiments, ciprofloxacin, doxycycline, and/or penicillin can beadministered. One of skill in the art will understand that severalantibiotics (including, but not limited to ciprofloxacin, doxycycline,and penicillin) can be combined with one or more anti-anthrax antibodiesto exert a preventive and/or therapeutic effect. The specificmethodology and results of an experiment conducted to confirm thecombined effects of antibody therapy and an exemplary antibiotic(ciprofloxacin) are described in detail as follows.

Methods: Hartley guinea pigs (250-300 g; n=9/group) and Swiss-Webstermice (25-30 g; n=10/group) were challenged by nasal instillation with 5LD₅₀ Bacillus anthracis spores 24 hours prior to twice dailysubcutaneous injections of ciprofloxacin for 6 days and/or a singleintraperitoneal injection of anti-PA mAb (AVP-21D9). Animal survival wasmonitored.

Results: Control animals challenged with anthrax spores died within 7days. AVP-21D9 provided protection and delayed the mean time of death;however, animals often succumbed to infection over the following weeks.The ED₅₀ dose of ciprofloxacin (3 mg/day, 6 days) protected the animalsover a 20-day period; drug toxicity was noted with a dose of 5.4 mg/day.When AVP-21D9 (1.5-15 mg) was combined with a low, non-protective doseof ciprofloxacin (1.12 mg/day), we observed synergistic protection ofthe animals for 30 days; however, higher doses of ciprofloxacin wereproportionately less effective. Similarly, Swiss-Webster mice werechallenged with 5 LD₅₀ Bacillus anthracis Ames. Ciprofloxacin (0.9mg/day) combined with 500 μg AVP-21D9 protected 100% of the animals formore than 30 days, while ciprofloxacin alone protected 60% and 21D9alone protected 40%. All survivors were re challenged with 5LD₅₀ valuesof Bacillus anthracis Ames spores by nasal instillation.

In view of the above results, it is believed that due to theasynchronous and delayed germination of the inhaled anthrax spores,prophylactic treatment of exposed individuals, in some embodiments, mayrequire prolonged administration of antibiotics. Thus, in someembodiments of the invention, a formulation and method to achievesynergistic protection of guinea pigs and mice against anthraxcomprising human mAb to PA and ciprofloxacin is provided. In oneembodiment, levels of ciprofloxacin used in combination therapy will beless than that needed if ciprofloxacin was used alone. Thisantibody/antibiotic combination may be beneficial in the clinical careof patients exposed to Bacillus anthracis spores.

Example 16 Mechanism of Action for Anti-Anthrax Antibodies

Bacillus anthracis uses two distinct strategies to evade immunesurveillance, thus facilitating dissemination throughout the body and arapid rise in bacteremia. Initially, upon exposure to spores, thecapsule composed of poly-D-glutamic acid provides a physical barrier tocircumvent phagocytosis. Secondly, via the concerted effect of threeproteins, PA, LF, and EF the immune response against the invadingbacteria is compromised. PA is a 83 kD protein that binds to tumorendothelial marker-8 (TEM-8) or human capillary morphogenesis protein-2(CMG-2), collectively called the anthrax toxin receptors (ATRs). Thereceptors are found on both macrophages and endothelial cells. LF is aprotease that inhibits mitogen-activated protein kinase-kinase, whichreduces the cytokine production by macrophages and ultimately leads tocell death. EF is an adenylate cyclase that generates cyclic AMP ineukaryotic cells and impairs the ability of neutrophils to engulfbacteria.

Although not wishing to be bound by this theory, it is believed that thetoxic effects of anthrax are initiated by PA. It is believed that PA83initially adheres to the membrane of the host cell, where it is thencleaved by a membrane-bound furin or furin-like protease which cleavesPA83 into two segments, PA63 and PA20. PA63, which is membrane-bound,assembles in groups of seven monomers (sometimes called a heptamer, ormore broadly a multimer or oligomer), thereby forming a heptamericchannel. The haptamer binds EF and LF, and allows EF and LF to enter thehost cell to exert their toxic effects.

Although previous researchers have attempted to prevent the binding ofEF and LF to PA63, the inventors believe that this is the first reportof a composition and method that prevents anthrax toxicity by preventingthe assembly of the PA63 heptamer.

The specific methodology and results of an experiment conducted toevaluate the mechanism of action of anti-anthrax toxins is describedbelow.

Materials and Methods: The binding of the monoclonal antibodies todistinct or overlapping epitopes on PA83 was determined by surfaceplasmon resonance. Anti-human IgG capture antibody (goat anti-human Fcgamma specific, Jackson ImmunoResearch) was coupled to a CM-5 chipthrough standard amine chemistry using an immobilization guide provideby the Biacore (Biacore Inc, NJ), whereby a response unit (RU) value of10,000 units was approached. The first human monoclonal antibody wasapplied, followed by pooled human (non-immune) IgG blocking antibody,PA83 (PA83, and LF both purchased from List Biological Labs, CA)followed sequentially with the second and third monoclonal antibodies.The order in which the antibodies were applied was changed in subsequentruns to cover all permutations with a vast excess of human polyclonalIgG blocking between steps. The test reagents (PA, MAbs) were applied at20 μg/ml in HBS-EP buffer provide by Biacore, the blocking antibodyhuman IgG/K1 (Sigma Co, MO) was used at 40 μg/ml. The resulting bindingdata is presented in FIG. 15.

To map the antibody recognition to distinct portion of the PA83, westernblot analysis was undertaken. In FIG. 16A, a schematic of fragments ofPA83 generated by trypsin and chymotrypsin digests based on thesequences and mapping studies previously described is shown (Welkos S L,Lowe J R, Eden-McCutchan F, Vodkin M, Leppla S H, Schmidt J J, Sequenceand analysis of the DNA encoding protective antigen of Bacillusanthracis, Gene 69 (1988), 2:287-300; Novak J M, Stein M P, Little S F,Leppla S H, Friedlander A M, Functional characterization ofprotease-treated Bacillus anthracis protective antigen, J Biol Chem 267(1992), 24:17186-93, both herein incorporated by reference). IntactPA83, trypsin, chymotrypsin and a combination of trypsin andchymotrypsin generated PA fragments were probed with human monoclonalantibodies AVP-1C6, AVP-22G12 and AVP-21D9 in a western blot (FIG. 16B).

To investigate whether the antibody bound to PA83 blocked subsequentprocessing, PA83 was pre-incubated with antibodies and treated withtrypsin. The resulting mixtures were pulled down with protein A andanalysed by SDS-PAGE and Coomassie staining (FIG. 16C).

The role of antibodies in inhibiting the binding of lethal factor toPA63 oligomer was again investigated by surface plasmon resonance. PA63(PA63 oligomer, List Laboratories, CA) was immobilized on a BiaCore CM5chip, essentially as described above, antibody captured and lethalfactor applied. The binding events were monitored and are presented in asensogram (FIG. 17A). Also, the role of antibodies in blocking bindingof the anthrax toxin PA83 to its receptor was investigated in a similarmanner. Anti-human Fc gamma was conjugated to the CM5 chip, humanmonoclonal anti-PA antibody was captured, PA83 was bound and the solubleanthrax toxin receptor was applied (plasmid encoding full-length ATR waskindly provided by Dr. Ken Bradley UCLA). Again each binding event wasmonitored by surface plasmon resonance (FIG. 17B).

Finally, to determine if the antibodies inhibited the formation of PA63heptamer, equimolar amounts of PA83 (0.25 nmol) and anti-PA antibody(0.25 nmol) were mixed in 70 μl of PBS. After 30 minutes incubation atroom temperature, the mixture was transferred to 4° C. and 10 μl ofice-cold trypsin (50 μg/ml) was added for 5 minutes. Trypsin wasinactivated by the addition of 5 μl trypsin and chymotrypsin inhibitor(10 mg/ml). Citric phosphate buffer (115 μl of 0.1M, pH 5.0) was thenadded to bring the pH to 5.0 to facilitate PA63 oligomerization. SDSloading buffer was added and the mixtures placed on boiling water for 10minutes. Polypeptides were separated in a 10% Bis-Tris gel underreducing condition. Protein bands were visualized by Coomassie bluestaining (FIG. 18).

Results: Sequential binding of anti-PA antibodies to PA83 indicated thatthey bound to distinct non-overlapping epitopes (FIG. 15). Western blotanalysis of intact PA 83 indicated all three antibodies recognizedlinear epitopes on PA83 (FIGS. 16A and 16B). AVP-21D9 and AVP-1C6 mappedto the carboxyl domain PA47 generated by chymotrypsin digestion. Thebinding of AVP-22G12 mapped to the chymotrypsin generated fragment PA37which contains the natural furin cleavage site. Treating PA83 withtrypsin abolished AVP-22G12 binding in the western blot. Initially, thissuggested that AVP-22G12 itself might act by inhibiting the cleavage ofPA83 to PA63. To test this hypothesis, PA83 was pre-incubated withantibody prior to trypsin addition, the resulting mixture was analyzed.Surprisingly, AVP-22G12 bound to PA83 permits cleavage by trypsin,implying that it binds close to (and possibly spans) the accessiblecleavage site (FIG. 16C).

To determine whether the antibodies efficacy was in part due toinhibiting LF binding to PA63 oligomer, the interactions between PA63oligomer, antibody and LF were investigated. AVP-22G12 did not bind topreformed PA63 oligomer, thus by default did not appear to compete forEF/LF binding; though AVP-21D9 had very weak binding (possibly due tothe presence of a small amount of PA63 monomer) and AVP-1C6 bound to thePA 63 oligomer, neither inhibited LF binding (FIG. 17A). All theantibodies bound PA83, which subsequently bound soluble ATR (sATR) (FIG.17B). However partial inhibition of sATR binding was observed on AVP-1C6captured PA83, hinting at a possible mode of action.

In this particular example, AVP-21D9 and AVP-22G12 did not inhibit PA83and sATR interaction, nor did they appear to prevent subsequentprocessing to PA63 or the binding of LF to PA 63 oligomer. Yet,paradoxically they were the two most potent inhibitors of anthrax lethaltoxin (PA/LF) in vitro and in vivo in rats. AVP-22G12 bound to nativePA83, denatured PA83 and PA37, but not to the preformed heptamer ormonomer PA63. Cleavage of PA83 to PA63 and PA20 completely abolished thebinding of AVP-22G12. However, if PA83 was bound to the antibody andsubsequently cleaved by trypsin, the resulting PA63-PA20 antibodycomplex remains bound (FIG. 16B and FIG. 16C). These observationsimplied that at least for AVP-22G12 the step of toxin neutralizationprobably occurred prior to heptamer assembly. In natural exposure toanthrax, upon cleavage of PA83 to PA63 and the release of PA20, the PA63spontaneously forms a heptamer.

An oligomer of PA63 can be formed in vitro by treating PA83 with trypsinand adjusting the pH 5.0. Once formed it is stable in the presence ofsodium dodecyl sulphate (SDS) as shown in lane 1 of FIG. 18. Antibodybound PA83 was cleaved by trypsin to mimic the natural furin likeprotease, to generate PA63-PA20 and pH was adjusted to 5.0 to facilitateheptamer assembly. The mixtures were examined by SDS-PAGE. In theabsence of anti-PA antibody (Lane 1) or in the presence of an isotypematched control antibody (Lane 5), the PA63 oligomer formed readily.Both AVP-22G12 and AVP-21D9 (Lanes 2 and 3) completely inhibitedheptamer formation (FIG. 18). Since western blot analysis had shown thatthe two antibodies bind to distinct regions of PA83, the antibodiesprevented the oligomer formation via distinct mechanisms. It wasdemonstrated that AVP-22G12 binds to a linear epitope on PA83 thatpossibly spans PA63 and PA20 cleavage site, but still permits access toprotease site and the clipped molecule retains PA20. It is plausiblethat the retention of PA20 on the antibody-antigen complex may hinderthe subsequent heptamer formation. Whereas AVP-21D9 bound to a distallinear epitope within the PA47 polypeptide and prevented PA oligomerformation possibly by masking potential assembly interfaces. It haspreviously been demonstrated that correct pore assembly is needed tofacilitate LF and EF entry into cells, thus blocking this portalmolecule effectively protects against the effects of both EF and LFanthrax toxin(s).

Thus, in view of the above results, it is believed that, in someembodiments, fully human antibodies generated in response to AVAvaccination neutralize anthrax exotoxin PA by interfering with and/orpreventing PA63 oligomer assembly.

Antibodies to Anthrax Made by Other Methods: In several embodiments ofthe present invention, a composition and method to treat and/or preventanthrax that prevents PA63 oligomer assembly is provided. The phrase“prevents PA63 assembly,” as used herein, shall be given its ordinarymeaning and shall also mean partially, substantially, or fullyinhibiting, interfering with, and/or disrupting the assembly of PA63into an oligomer. In one embodiment, the composition used to preventPA63 heptamer assembly is a binding agent, such as the anti-anthraxantibodies generated by the methods of the present invention. Forexample, in one embodiment, the antibody is a fully human monoclonalantibody generated using immuno-compromised mice. In other embodiments,however, antibodies created by the engineering of bacteriophages todisplay human monoclonal antibodies on their surface are used. In yetother embodiments, the composition comprises a mouse monoclonalantibody. In other embodiments, polyclonal antibodies are used. In yetother embodiments, humanized or chimeric antibodies are used. Methods ofmaking the antibodies described above (e.g., making humanizedantibodies, non-human monoclonal antibodies, and polyclonal antibodies)are well-known in the art.

In one embodiment, a composition and method for treating a mammalexposed to anthrax, or passively immunizing a mammal pre-exposure, isprovided. In one embodiment, the method comprises administering abinding agent to a mammal, wherein the binding agent prevents theassembly of a PA63 heptamer. By preventing the assembly of the PA63oligomer, the binding agent, in some embodiments, inhibits transport ofEF and/or LF into a mammalian host cell, thereby protecting the mammalfrom the toxic effects of anthrax. As discussed above, the binding agentcan be a monoclonal or polyclonal antibody. The binding agent can befully human, humanized, or non-human. Thus, any binding agent thatprevents, or otherwise interferes with the assembly of a PA63 oligomercan be used according to an embodiment of the current invention. Indeed,the binding agent need not be an antibody. For example, small molecules,such as peptides, that can bind to a site on the PA63 molecule, orotherwise prevent the assembly of the PA63 heptamer, can also be used(Bachhawat-Sikder, K., and Kodadek, T. (2003). Mixed element captureagents (MECAs): A simple strategy for the construction of synthetic,high affinity protein capture ligands. J Amer Chem Soc 125, 13995-14004,herein incorporated by reference).

FIG. 20 shows PA83 detection data in rat serum. In one example, purifiedPA83 was injected intravenously into rats at time t=0 and blood sampleswere taken every 10 minutes. Blood was collected in tubes containingheparin and spun immediately to separate the plasma from the cells. Thesamples were used in an ELISA assay as following. ELISA plates werecoated overnight with 5 μg/ml AVP-1C6, which serves as capture antibody.The plates were washed and blocked, and serum samples were added for 30minutes. The presence of PA83 in the samples was detected usingbiotinylated-polyclonal goat anti-PA (List Laboratories) andHRP-conjugated Avidin, followed by chromogenic detection. Theconcentration was calculated from a standard curve using PA83 at definedconcentrations. Other monoclonal antibodies described herein could alsobe used instead of polyclonal serum. The methods and data show anexample of an assay that is capable of detecting PA levels in serum.Such an assay can be used in one or more kits according to severalembodiments of the invention. Kits, in some embodiments, can be used todetermine the severity of anthrax exposure and/or the length of timefrom anthrax exposure. For example, by determining the levels of anthraxcomponents or metabolites in serum (or other biological samples), a kitmay be useful in determining the severity and type of anthrax infection.

In some embodiments, the binding agent prevents assembly of the PAheptamer by binding to a site on the PA63 molecule. In otherembodiments, the binding agent prevents assembly of the PA heptamer bybinding to a site on the PA20 molecule. In further embodiments, thebinding agent prevents assembly of the PA heptamer by binding to a siteon the PA83 molecule. In yet other embodiments, the binding agentprevents assembly of the PA heptamer by binding to a non-PA moleculethat is needed for PA assembly.

Although several different binding agents can be used in accordance withcertain embodiments of the present invention, antibodies (as opposed tonon-antibody agents) may be particularly advantageous because antibodiesmay bind with greater affinity and specificity to the desired epitope,thus preventing the assembly of PA63 more effectively. Further, fullyhuman antibodies are especially advantageous because fully humanantibodies may exert fewer side-effects in the body, and are likely tobe eliminated less rapidly from the body, thereby reducing the frequencyand amount of dosing. Further, fully human antibodies may have higheraffinities and specificities. While a number of preferred embodiments ofthe invention and variations thereof have been described in detail,other modifications and methods of use will be readily apparent to thoseof skill in the art. Accordingly, it should be understood that variousapplications, modifications and substitutions may be made of equivalentswithout departing from the spirit of the invention or the scope of theclaims.

1-20. (canceled)
 21. An isolated antibody or antigen-binding fragmentthereof which specifically binds to the same protective antigen (PA)epitope as an anti-anthrax toxin monoclonal antibody, wherein saidanti-anthrax toxin monoclonal antibody is 21D9.
 22. (canceled)
 23. Theisolated antibody or fragment thereof of claim 21, wherein the heavychain variable region (VH) of said antibody or fragment thereofcomprises at least one heavy chain complementarity determining region(VH-CDR) which is identical, except for variations or modifications thatdo not alter specific binding activity of said antibody or fragmentthereof to anthrax toxin, to a VH-CDR selected from the group consistingof VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NO:
 2. 24-27. (canceled) 28.The isolated antibody or fragment thereof of claim 21, wherein the lightchain variable region (VL) of said antibody or fragment thereofcomprises at least one light chain complementarity determining region(VL-CDR) which is identical, except for variations or modifications thatdo not alter specific binding activity of said antibody or fragmentthereof to anthrax toxin, to a VL-CDR selected from the VL-CDR1,VL-CDR2, and VL-CDR3 of SEQ ID NO:
 4. 29-32. (canceled)
 33. The isolatedantibody or fragment thereof of claim 23, wherein the VH of saidantibody or fragment thereof comprises a VH-CDR3 amino acid sequencewhich is identical, except for variations or modifications that do notalter specific binding activity of said antibody or fragment thereof toanthrax toxin, to amino acids 99-110 of SEQ ID NO:
 2. 34. The isolatedantibody or fragment thereof of claim 33, wherein the VH-CDR3 amino acidsequence comprises amino acids 99-110 of SEQ ID NO:
 2. 35. The isolatedantibody or fragment thereof of claim 28, wherein the VL of saidantibody or fragment thereof comprises a VL-CDR3 amino acid sequencewhich is identical, except for variations or modifications that do notalter specific binding activity of said antibody or fragment thereof toanthrax toxin, to amino acids 89-96 of SEQ ID NO:
 4. 36. The isolatedantibody or fragment thereof of claim 35, wherein the VL-CDR3 amino acidsequence comprises amino acids 89-96 of SEQ ID NO:
 4. 37. The isolatedantibody or fragment thereof of claim 21, wherein the VH of saidantibody or fragment thereof comprises VH-CDR1, VH-CDR2, and VH-CDR3amino acid sequences which are identical, except for variations ormodifications that do not alter specific binding activity of saidantibody or fragment thereof to anthrax toxin, to amino acids 31-35,50-66, and 99-110 of SEQ ID NO: 2, respectively.
 38. The isolatedantibody or fragment thereof of claim 37, wherein the VH-CDR1 amino acidsequence comprises amino acids 31-35 of SEQ ID NO:
 2. 39. The isolatedantibody or fragment thereof of claim 37, wherein the VH-CDR2 amino acidsequence comprises amino acids 50-66 of SEQ ID NO:
 2. 40. The isolatedantibody or fragment thereof of claim 37, wherein the VH-CDR3 amino acidsequence comprises amino acids 99-110 of SEQ ID NO:
 2. 41. The isolatedantibody or fragment thereof of claim 37, wherein the VH-CDR1, VH-CDR2,and VH-CDR3 amino acid sequences comprise amino acids 31-35, 50-66, and99-110 of SEQ ID NO: 2, respectively.
 42. The isolated antibody orfragment thereof of claim 21, wherein the VL of said antibody orfragment thereof comprises VL-CDR1, VL-CDR2, and VL-CDR3 amino acidsequences which are identical, except for variations or modificationsthat do not alter specific binding activity of said antibody or fragmentthereof to anthrax toxin, to amino acids 24-34, 50-56, and 89-96 of SEQID NO: 4, respectively.
 43. The isolated antibody or fragment thereof ofclaim 42, wherein the VL-CDR1 amino acid sequence comprises amino acids24-34 of SEQ ID NO:
 4. 44. The isolated antibody or fragment thereof ofclaim 42, wherein the VL-CDR2 amino acid sequence comprises amino acids50-56 of SEQ ID NO:
 4. 45. The isolated antibody or fragment thereof ofclaim 42, wherein the VL-CDR3 amino acid sequence comprises amino acids89-96 of SEQ ID NO:
 4. 46. The isolated antibody or fragment thereof ofclaim 42, wherein the VL-CDR1, VH-CDR2, and VH-CDR3 amino acid sequencescomprise amino acids 24-34, 50-56, and 89-96 of SEQ ID NO: 4,respectively. 47-63. (canceled)
 64. An isolated polynucleotidecomprising a nucleic acid sequence which encodes an antibody orantigen-binding fragment thereof, wherein said antibody orantigen-binding fragment thereof comprises at least one VH-CDR of a VHpolypeptide which specifically binds to the same protective antigen (PA)epitope as an anti-anthrax toxin monoclonal antibody, wherein saidanti-anthrax toxin monoclonal antibody is 21D9; wherein said VH-CDR isencoded by a polynucleotide sequence that is identical, except forvariations or modifications due to degeneracy in the genetic code, to apolynucleotide sequence selected from the group consisting of: VH-CDR1,VH-CDR2, and VH-CDR3 of SEQ ID NO:
 1. 65. An isolated polynucleotidecomprising a nucleic acid sequence which encodes an antibody orantigen-binding fragment thereof, wherein said antibody orantigen-binding fragment thereof comprises at least one VL-CDR of a VLpolypeptide which specifically binds to the same protective antigen (PA)epitope as an anti-anthrax toxin monoclonal antibody, wherein saidanti-anthrax toxin monoclonal antibody is 21D9; wherein said VL-CDR isencoded by a polynucleotide sequence that is identical, except forvariations or modifications due to degeneracy in the genetic code, to apolynucleotide sequence selected from the group consisting of: VL-CDR1,VL-CDR2, and VL-CDR3 of SEQ ID NO:
 3. 66. (canceled)
 67. (canceled) 68.A polypeptide encoded by the polynucleotide of claim
 64. 69. A vectorcomprising the polynucleotide of claim
 64. 70. A host cell comprisingthe vector of claim
 69. 71. A method of producing an antibody orfragment thereof which specifically binds PA, comprising culturing thehost cell of claim 70, and recovering said antibody or fragment thereof.72. An antibody or fragment thereof recovered by the method of claim 71.73. A composition comprising a first antibody or fragment thereof ofclaim 21 and one or more additional therapeutic agents, wherein saidadditional therapeutic agent is selected from the group consisting of avaccine, an antibiotic, and a second antibody or fragment thereof.74-79. (canceled)
 80. An isolated antibody or antigen-binding fragmentthereof which specifically binds to a protective antigen (PA) andinhibits PA63 assembly, wherein said isolated antibody orantigen-binding fragment thereof binds the distal linear epitope withinPA47 polypeptide of PA83.
 81. The isolated antibody or antigen-bindingfragment thereof of claim 80, wherein said isolated antibody orantigen-binding fragment has a binding affinity of about 1×10⁻¹¹ toabout 1×10⁻¹⁰ M.
 82. A method of preventing PA63 assembly in a mammalcomprising administration of the isolated antibody or antigen-bindingfragment thereof of claim 80 to said mammal thereby preventing PA63assembly.
 83. A polypeptide encoded by the polynucleotide of claims 65.84. A vector comprising the polynucleotide of
 65. 85. A host cellcomprising the vector of claim
 84. 86. A method of producing an antibodyor fragment thereof which specifically binds PA, comprising culturingthe host cell of claim 85, and recovering said antibody or fragmentthereof.
 87. An antibody or fragment thereof recovered by the method ofclaim 86.